Multivalent influenza vaccine

Abstract

An octavalent influenza vaccine is provided, comprising eight messenger RNAs (mRNAs), each of which comprises an open reading frame encoding a different influenza antigen. Lipid nanoparticles (LNPs) for delivering said mRNAs are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 212,523, filed June 18, 2021; U.S. Provisional Application No. 63 / 276,243, filed November 5, 2021; International Application PCT / US2021 / 058250, filed November 5, 2021; and European Patent Application Publication No. 21315198.8, filed October 13, 2021, the contents of each of which are incorporated by reference in their entirety for all purposes. [Background technology]

[0002] Messenger RNA (mRNA)-based vaccines offer a promising alternative to traditional subunit vaccines that contain antigenic proteins derived from pathogens. The antigenic proteins are usually produced recombinantly and require bacterial fermentation and / or cell culture, as well as complex purification. mRNA-based vaccines allow de novo expression of complex antigens in vaccinated subjects, which in turn allows for proper post-translational modification and presentation of the antigen in its native form. Unlike traditional techniques, the production of mRNA vaccines does not require complex and costly bacterial fermentation, tissue culture, and purification methods. Furthermore, once established, manufacturing methods for mRNA vaccines are available for a variety of antigens, allowing for the rapid development and distribution of mRNA vaccines. Moreover, mRNA vaccines are inherently safe delivery vectors, since they only express antigens transiently and do not integrate into the host genome. Because the antigens encoded by the mRNA are produced in vivo in the vaccinated individual, mRNA vaccines are particularly effective in eliciting both humoral and T cell-mediated immunity. Summary of the Invention [Problem to be solved by the invention]

[0003] Current licensed influenza vaccines are live attenuated or inactivated influenza vaccines, and are often produced in cell culture or eggs. In addition, multiple influenza strains may circulate within the population every year, making it difficult for a single influenza vaccine to provide robust protection against multiple strains. Therefore, there is a need for mRNA-based influenza vaccines, including multivalent mRNA-based influenza vaccines that target multiple influenza strains. [Means for solving the problem]

[0004] The present disclosure provides an influenza vaccine composition that includes eight messenger RNAs (mRNAs), each of which contains an open reading frame (ORF) encoding a different influenza antigen.

[0005] In certain embodiments, the composition comprises eight mRNAs encoding (i) one or more hemagglutinin (HA) antigens, (ii) one or more neuraminidase (NA) antigens, or (iii) at least one HA antigen and at least one NA antigen.

[0006] In certain embodiments, the composition comprises one or more mRNAs encoding antigens of influenza A, B and / or C viruses.

[0007] In certain embodiments, the antigens are HA and / or NA antigens of influenza A and influenza B viruses.

[0008] In certain embodiments, the NA antigen of the influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11.

[0009] In certain embodiments, the influenza B virus HA and NA antigens are from influenza B / Yamagata or influenza B / Victoria lineages.

[0010] In certain embodiments, the HA and NA antigens are selected from the group consisting of H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H10N7 subtypes and / or B / Yamagata and B / Victoria lineages.

[0011] In certain embodiments, the composition comprises one mRNA encoding an H3 HA antigen, one mRNA encoding an H1 HA antigen, one mRNA encoding an HA antigen from the influenza B / Yamagata lineage, and one mRNA encoding an HA antigen from the influenza B / Victoria lineage.

[0012] In certain embodiments, the composition comprises one mRNA encoding an H3 HA antigen, one mRNA encoding an N2 NA antigen, one mRNA encoding an H1 HA antigen, one mRNA encoding an N1 NA antigen, one mRNA encoding an HA antigen from influenza B / Yamagata lineage, one mRNA encoding an NA antigen from influenza B / Yamagata lineage, one mRNA encoding an HA antigen from influenza B / Victoria lineage, and one mRNA encoding an NA antigen from influenza B / Victoria lineage.

[0013] In certain embodiments, the ORF is codon optimized.

[0014] In certain embodiments, an mRNA molecule comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and at least one polyadenylation (poly(A)) sequence.

[0015] In certain embodiments, the mRNA comprises at least one chemical modification.

[0016] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0017] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0018] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0019] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0020] In certain embodiments, the chemical modification is N1-methylpseudouridine.

[0021] In certain embodiments, the mRNA is formulated in a lipid nanoparticle (LNP).

[0022] In certain embodiments, the LNP comprises at least one cationic lipid.

[0023] In certain embodiments, the cationic lipid is biodegradable. In certain embodiments, the cationic lipid is not biodegradable.

[0024] In certain embodiments, the cationic lipid is cleavable. In certain embodiments, the cationic lipid is not cleavable.

[0025] In certain embodiments, the cationic lipid is selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14.

[0026] In certain embodiments, the LNPs further comprise polyethylene glycol (PEG)-conjugated (pegylated) lipids, cholesterol-based lipids, and helper lipids.

[0027] In certain embodiments, the LNP comprises: cationic lipids at molar ratios of 35%–55%; polyethylene glycol (PEG)-conjugated (PEGylated) lipids at molar ratios of 0.25% to 2.75%; Cholesterol-based lipids in molar ratios between 20% and 45%; and Helper lipids at molar ratios of 5% to 35% All molar ratios are based on the total lipid content of the LNP.

[0028] In certain embodiments, the LNP comprises: cationic lipid at a molar ratio of 40%; PEGylated lipid at a molar ratio of 1.5%; Cholesterol-based lipids at a molar ratio of 28.5%; and Helper lipid at 30% molar ratio include.

[0029] In certain embodiments, the pegylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0030] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0031] In certain embodiments, the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0032] In certain embodiments, the LNP comprises: a cationic lipid selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30% include.

[0033] In certain embodiments, the LNPs have an average diameter between 30 nm and 200 nm, hi certain embodiments, the LNPs have an average diameter between 80 nm and 150 nm.

[0034] In certain embodiments, the influenza vaccine composition comprises between 1 mg / mL and 10 mg / mL of LNP.

[0035] In certain embodiments, the LNPs comprise between 1 and 20 mRNA molecules, hi certain embodiments, the LNPs comprise between 5 and 10 or between 6 and 8 mRNA molecules.

[0036] In certain embodiments, the LNP comprises two or more mRNAs, each encoding a different influenza antigen.

[0037] In certain embodiments, the composition comprises eight LNPs, each LNP comprising an mRNA encoding a different influenza antigen.

[0038] In certain embodiments, the composition is formulated for intramuscular injection.

[0039] In certain embodiments, the composition comprises phosphate buffered saline.

[0040] In one aspect, the disclosure provides a method of eliciting an immune response in a subject in need thereof, comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of an influenza vaccine composition as described above.

[0041] In one aspect, the disclosure provides a method of preventing influenza infection or reducing one or more symptoms of influenza infection comprising administering to a subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of an influenza vaccine composition as described above.

[0042] In certain embodiments, the influenza vaccine composition elicits an immune response against one or more seasonal and / or pandemic influenza strains.

[0043] In certain embodiments, the methods include administering to the subject one or more doses of an influenza vaccine composition, each dose comprising from about 1 μg to about 250 μg of mRNA.

[0044] In certain embodiments, the methods include administering to a subject one or more doses of an influenza vaccine composition, each dose comprising about 2.5, 5, 15, 45, or 135 μg of mRNA.

[0045] In certain embodiments, the methods involve administering to the subject two doses of an influenza vaccine composition, spaced 2-6 weeks apart, optionally 4 weeks apart.

[0046] In another aspect, the disclosure provides the use of the influenza vaccine composition described above for the manufacture of a medicament for use in treating a subject in need thereof.

[0047] In certain embodiments, the influenza vaccine composition is intended for use in treating a subject in need of treatment.

[0048] In another aspect, the disclosure provides a kit comprising a container containing a single-use or multi-use dosage of the composition, optionally the container is a vial or a pre-filled syringe or injector.

[0049] In certain embodiments, the influenza antigen comprises an influenza virus HA antigen and / or an influenza virus NA antigen having a molecular sequence identified or designed from a machine learning model. [Brief description of the drawings]

[0050] [Figure 1A]FIG. 1A is a pair of graphs showing expression of human erythropoietin (hEPO) in mice treated with various LNP formulations of hEPO mRNA. Panel a): LNP formulations "Lipid A" and "Lipid B" compared to MC3. Bars represent the mean and standard deviation. Panel b): Formulations made with cationic lipid OF-02. PEG: DMG-PEG2000. Cholest: Cholesterol. "Lipid A": LNP composition containing OF-02, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30, unless otherwise indicated. "Lipid B": LNP composition containing cKK-E10, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 1B] FIG. 1B is a pair of graphs showing expression of hEPO in mice and non-human primates (NHPs) using LNP formulations Lipid A and Lipid B. [Figure 2A]Figures 2A and 2B are a pair of graphs showing that lipid A and lipid B LNP formulations with mRNA encoding hemagglutinin (HA) of strain A / California / 7 / 2009(H1N1)(CA09) induced robust functional antibodies (Figure 2A) and protected mice from death or emaciation (>20%) when challenged with a pandemic strain of influenza virus (Figure 2B). Hemagglutinin inhibition (HAI) titers are reported as log10 for serum samples taken on study days 0, 14, 28, 42, 56, 92, and 107. Bars are geometric means and geometric standard deviations. Daily body weights were measured after intranasal challenge (day 93) with 4LD50 of A / Belgium / 2009(H1N1)(Belgium09). Body weights are presented as percentage of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting body weight and in all mice 14 days post-infection (day 107). rHA: recombinant hemagglutinin. AF03: oil in water emulsion adjuvant. Diluent = PBS. LLOQ = lower limit of quantification. 1 / 40 = 1 / 40 minimum target, which is the HAI antibody titer associated with a 50% reduction in risk of influenza infection or disease in healthy adults (Coudeville et al., BMC Med Res Methodol. (2010) 10:18). Dashed line in Figure 2B = 20% body weight loss clipped with respect to body weight on the day of challenge. [Figure 2B]Figures 2A and 2B are a pair of graphs showing that lipid A and lipid B LNP formulations with mRNA encoding hemagglutinin (HA) of strain A / California / 7 / 2009(H1N1)(CA09) induced robust functional antibodies (Figure 2A) and protected mice from death or emaciation (>20%) when challenged with a pandemic strain of influenza virus (Figure 2B). Hemagglutinin inhibition (HAI) titers are reported as log10 for serum samples taken on study days 0, 14, 28, 42, 56, 92, and 107. Bars are geometric means and geometric standard deviations. Daily body weights were measured after intranasal challenge (day 93) with 4LD50 of A / Belgium / 2009(H1N1)(Belgium09). Body weights are presented as percentage of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting body weight and in all mice 14 days post-infection (day 107). rHA: recombinant hemagglutinin. AF03: oil in water emulsion adjuvant. Diluent = PBS. LLOQ = lower limit of quantification. 1 / 40 = 1 / 40 minimum target, which is the HAI antibody titer associated with a 50% reduction in risk of influenza infection or disease in healthy adults (Coudeville et al., BMC Med Res Methodol. (2010) 10:18). Dashed line in Figure 2B = 20% body weight loss clipped with respect to body weight on the day of challenge. [Figure 3A]Figures 3A and 3B are a pair of graphs showing that A / Michigan / 45 / 2015 (Mich15) neuraminidase (NA) mRNA formulated with lipid A LNPs induced robust functional antibodies (Figure 3A) and protected mice from weight loss and death when challenged with a pandemic strain of influenza virus (Figure 3B). Neuraminidase inhibition (NAI) titers are reported as log10 for serum samples taken on study days 14, 28, 42, 56, 88, and 114. Daily body weights were measured after intranasal challenge with 4LD50 of Belgium09 (day 89 for the single dose group or day 117 for the two dose group). Body weights are presented as percentage of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting body weight and in all mice 14 days post-infection (day 103 for the single dose group or day 131 for the double dose group). Bars are mean and standard deviation. Upper dashed line in Fig. 3A = upper limit of quantification. Lower dashed line in Fig. 3A = lower limit of quantification. Dashed line in Fig. 3B = 20% body weight loss clipped with respect to body weight on the day of challenge. Administered mRNA: 0.4 or 0.016 μg of mRNA encoding Mich15 NA. Control: 0.6 μg of mRNA encoding hEPO or diluent (PBS). [Figure 3B]Figures 3A and 3B are a pair of graphs showing that A / Michigan / 45 / 2015 (Mich15) neuraminidase (NA) mRNA formulated with lipid A LNPs induced robust functional antibodies (Figure 3A) and protected mice from weight loss and death when challenged with a pandemic strain of influenza virus (Figure 3B). Neuraminidase inhibition (NAI) titers are reported as log10 for serum samples taken on study days 14, 28, 42, 56, 88, and 114. Daily body weights were measured after intranasal challenge with 4LD50 of Belgium09 (day 89 for the single dose group or day 117 for the two dose group). Body weights are presented as percentage of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting body weight and in all mice 14 days post-infection (day 103 for the single dose group or day 131 for the double dose group). Bars are mean and standard deviation. Upper dashed line in Fig. 3A = upper limit of quantification. Lower dashed line in Fig. 3A = lower limit of quantification. Dashed line in Fig. 3B = 20% body weight loss clipped with respect to body weight on the day of challenge. Administered mRNA: 0.4 or 0.016 μg of mRNA encoding Mich15 NA. Control: 0.6 μg of mRNA encoding hEPO or diluent (PBS). [Figure 4] Figure 4 is a graph showing that Lipid A and Lipid B LNP formulations (10 μg) with CA09 HA mRNA induced robust functional antibodies in cynomolgus macaques. HAI titers are reported as log2 for serum samples taken on study days 0, 14, 28, 42, and 56. [Figure 5A-1] Figures 5A-C show the MRT1400 mRNA encoding influenza virus A / Singapore / INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin. Figure 5A: Alignment of wild-type (WT) and codon-optimized genes (MRT10279) for the HA antigen. Figure 5B: Structure of the mRNA. Figure 5C: Sequence of the mRNA. [Figure 5A-2] Continued from Figure 5A-1. [Figure 5A-3] Continued from Figure 5A-2. [Figure 5B] Figures 5A-C show the MRT1400 mRNA encoding influenza virus A / Singapore / INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin. Figure 5A: Alignment of wild-type (WT) and codon-optimized genes (MRT10279) for the HA antigen. Figure 5B: Structure of the mRNA. Figure 5C: Sequence of the mRNA. [Figure 5C] Figures 5A-C show the MRT1400 mRNA encoding influenza virus A / Singapore / INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin. Figure 5A: Alignment of wild-type (WT) and codon-optimized genes (MRT10279) for the HA antigen. Figure 5B: Structure of the mRNA. Figure 5C: Sequence of the mRNA. [Figure 6] FIG. 6 is a pair of graphs showing that lipid A and lipid B LNP formulations with MRT1400 or NA mRNA induced robust functional antibodies in mice. The first injection was given on study day 0 and the second injection was given on study day 28. Left panel: HAI titers are reported as log10 for serum samples taken on study days 14, 28, 42, and 56. Right panel: NAI titers are reported as log10 for serum samples taken on study days 14, 28, 42, and 56. Bars are geometric mean and geometric standard deviation. Dashed line = lower limit of quantification. [Figure 7A] FIG. 7A is a graph showing that lipid A and lipid B LNP formulations with MRT1400 induced robust functional antibodies in NHPs. HAI titers are reported as log2 for serum samples taken on study days 0, 14, 28, 42, and 56. The first injection was given on study day 0 and the second injection was given on study day 28. Bars are the mean and standard deviation. Top dashed line = 1 / 40 minimum target. Bottom dashed line = lower limit of detection. [Figure 7B]Figures 7B and 7C are a pair of figures showing that lipid A LNP formulations containing MRT1400 mRNA (MRT5400) induced functional antibodies (Figure 7B) and robust ELISA titers (Figure 7C) in cynomolgus macaques at four dose levels of mRNA: 15, 45, 135 and 250 μg. HAI and ELISA titers are reported as log2 for serum samples taken on study days 0, 14, 28, 42, and 56. The first injection was given on study day 0 and the second injection was given on study day 28. Bars are mean and standard deviation. Dashed line = 1 / 40 minimum target. [Figure 7C] Figures 7B and 7C are a pair of figures showing that lipid A LNP formulations containing MRT1400 mRNA (MRT5400) induced functional antibodies (Figure 7B) and robust ELISA titers (Figure 7C) in cynomolgus macaques at four dose levels of mRNA: 15, 45, 135 and 250 μg. HAI and ELISA titers are reported as log2 for serum samples taken on study days 0, 14, 28, 42, and 56. The first injection was given on study day 0 and the second injection was given on study day 28. Bars are mean and standard deviation. Dashed line = 1 / 40 minimum target. [Figure 8A] Figures 8A and 8B are panels of graphs showing T cell cytokine responses of cynomolgus macaques after a second vaccination with lipid A LNP formulation MRT5400 in three dose level groups (250 μg, 135 μg, and 45 μg mRNA). IFN-γ and IL-13 induced by restimulation with recombinant HA (rHA) protein (left panel) or pooled peptides (right panel) were assessed in peripheral blood mononuclear cells (PMBCs) on day 42 by ELISPOT assay. The frequency of PBMC secreting IFN-γ (Figure 8A) or IL-13 (Figure 8B) was calculated as speckle forming cells (SFC) per million PBMCs. Each symbol represents an individual sample and the bars represent standard deviation. [Figure 8B]Figures 8A and 8B are panels of graphs showing T cell cytokine responses of cynomolgus macaques after a second vaccination with lipid A LNP formulation MRT5400 in three dose level groups (250 μg, 135 μg, and 45 μg mRNA). IFN-γ and IL-13 induced by restimulation with recombinant HA (rHA) protein (left panel) or pooled peptides (right panel) were assessed in peripheral blood mononuclear cells (PMBCs) on day 42 by ELISPOT assay. The frequency of PBMC secreting IFN-γ (Figure 8A) or IL-13 (Figure 8B) was calculated as speckle forming cells (SFC) per million PBMCs. Each symbol represents an individual sample and the bars represent standard deviation. [Figure 9A] Figure 9A is a pair of graphs showing that lipid A LNP formulations containing modified and unmodified CA09 HA mRNA were comparable as indicated by HAI titers in vaccinated mice. HAI titers are reported as log2 for serum samples taken on study days 14, 28, 42, and 56. The first injection was given on study day 0 and the second injection was given on study day 28. Bars are the mean and standard deviation. Top dashed line = 1 / 40 minimum target. Bottom dashed line = lower limit of quantification. [Figure 9B] Figure 9B is a pair of graphs showing that lipid A LNP formulations containing modified and unmodified CA09 HA mRNA were comparable in mice as indicated by ELISA titers. Total IgG ELISA titers are reported as log10 for serum samples taken on study days 14, 28, 42, and 56. The first injection was given on study day 0 and the second injection was given on study day 28. Dashed line = lower limit of quantitation. [Figure 10]Figures 10A and 10B are a pair of graphs showing that bivalent lipid A LNP formulations with CA09 HA mRNA and Sing16 HA mRNA induced robust functional antibodies as assessed by HAI titers (CA09 (Figure 10A) and Sing16 (Figure 10B)) in Balb / c mice at a dose of 0.4 μg total mRNA. 0.4 μg mRNA was administered as a co-encapsulated mRNA-LNP formulation or each HA mRNA was administered separately with 0.2 μg in each leg. Each HA mRNA was also co-encapsulated in the formulation along with a non-coding mRNA to control for total mRNA being packaged in the LNP. Dilution groups received mRNA-LNP dilution buffer. HAI titers are reported for serum samples taken on study days -2 (baseline), 14, 28, and 42. Figure 10B shows only study days -2 (baseline from pooled serum) and 42. The first injection was given on study day 0 and the second injection was given on study day 28. Bars are geometric mean and geometric standard deviation. Dashed line = lower limit of quantification. [Figure 11]Figure 11 shows the functional validation of mRNA-LNP formulations. Panel (a) is a graph showing the expression of firefly (FF) luciferase in BALB / c mice: A single dose of luciferase FF mRNA-LNP (5, 1, 0.1, 0.05 μg) was injected into mice (n=4) by IM route. Luciferin (3 mg) was injected at the time of whole-body animal imaging using IVIS Spectrum, Perkin Elmer, which records bioluminescence intensity. Images of whole-body animal mean luminescence were taken 6, 24, 48 and 72 hours after injection. The luminescence recorded for 1, 0.5, 0.1 and 0.05 μg dose administration of Luc mRNA-LNP is shown in the graph. Panel (b) shows whole-body animal images showing the total flux of luminescence from 6 to 72 hours. The total flux of luminescence in a group of mice (n=4) receiving 0.1 μg dose of FF-LNP is shown. Panel (c) shows expression of hEPO in BALB / c mice. A single dose of hEPO mRNA-LNP (0.1 μg) was injected by IM route in BALB / c mice. hEPO expression was quantified in serum using ELISA at 6 and 24 hours after administration. Bars represent the mean and standard deviation. Panel (d) shows expression of hEPO in NHPs. A single dose of hEPO mRNA-LNP (10 μg) was injected by IM route in cynomolgus macaques. hEPO expression was quantified in serum using ELISA at 6, 24, 48, 72, and 96 hours after administration. Bars represent the mean and standard deviation. [Figure 12] Figure 12 shows serological evaluation of HA mRNA-LNP vaccine in mice. BALB / c mice (n=8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of Cal09 HA mRNA-LNP or Sing16 HA mRNA-LNP. ELISA titers recorded for sera collected on days 14, 28, 42, and 56 against CA09 (Cal09) H1N1 influenza virus recombinant HA (left panel) and Sing16 H3N2 influenza virus recombinant HA (right panel) are shown. [Figure 13]Figure 13 shows serological evaluation of HA mRNA-LNP vaccine in mice. BALB / c mice (n=8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of CA09 HA mRNA-LNP or Sing16 HA mRNA-LNP. Log10 HAI titers recorded against CA09 H1N1 influenza virus (left panel) and Sing16 H3N2 influenza virus (right panel) are shown. [Figure 14] Figure 14 shows serological evaluation of NA mRNA-LNP vaccine in mice. BALB / c mice (n=8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP. Total IgG titers recorded for sera collected on days 0, 14, 28, 42, and 56 against Mich15 N1 influenza virus recombinant NA (left panel) and Sing16 N2 influenza virus recombinant NA (right panel) are shown. [Figure 15] Figure 15 shows serological evaluation of NA mRNA-LNP vaccines in mice. BALB / c mice (n=8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP. Log10NAI(ELLA) titers recorded for sera against Mich2015(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus (left panel) and Sing16(N2):A / Mallard / Sweden / 2002(H6) chimeric virus (right panel) are shown. [Figure 16]Figures 16A and 16B show the protective efficiency of CA09 HA mRNA-LNP vaccine in mice after lethal A / Belgium / 2009 H1N1 virus challenge. Mice (n=8) received two IM doses of CA09 HA mRNA-LNP (0.4 μg each) on days 0 and 28. Control animals received two IM doses of diluent on days 0 and 28. Figure 16A shows HAI titers reported as Log10 for serum samples taken on study days 0, 14, 28, 42, 56, 92, and 107. Figure 16B shows daily body weights after intranasal challenge on day 93 with 4 LD50 of A / Belgium / 2009 H1N1 strain. Body weights are presented as percentage of weight loss from the day of challenge. Individual lines represent each animal. [Figure 17] 17A-B show the protective efficacy of a single dose of unmodified Mich15 NA mRNA-LNP in mice following lethal A / Belgium / 2009 H1N1 virus challenge. Mice (n=16) were injected with 0.4 μg or 0.016 μg of Mich15 NA mRNA-LNP by IM route. Half of the mice received only one injection (1 dose) on study day 0, while the other half (2 doses) received two injections given on study day 0 and study day 28. Control animals received two IM doses of hEPO mRNA-LNP (0.6 μg) on ​​days 0 and 28. FIG. 17A shows that NAI titers are reported as Log10 for serum samples taken on study days 0, 14, 28, 42, 56, 88, and 114. Figure 17B shows daily body weight changes following intranasal challenge with 4LD50 of Belgium09 H1N1 on day 89 for the single dose group and on day 117 for the two dose group. Body weights are presented as percentage of weight loss from the day of challenge. Individual lines represent each animal. [Figure 18]Figure 18 shows serological evaluation of HA Sing16 HA mRNA-LNP vaccine in NHP. Cynomolgus macaques (n=6 per group) were injected twice, 4 weeks apart, with 15, 45 or 135 μg of Sing16 HA mRNA-LNP by IM route. Serum samples were collected on days -6, 14, 28, 42, and 56. Log10 IgG titers against recombinant HA protein of Sing16 virus are shown. [Figure 19] Figures 19A and 19B show serological evaluation of HA Sing16 HA mRNA-LNP vaccine in NHPs. Cynomolgus macaques (n=6 per group) were injected twice, 4 weeks apart, with 15, 45 or 135 μg of Sing16 HA mRNA-LNP by IM route. Serum samples were collected on days 0, 14, 28, 42, and 56. Log10 HAI titers (Figure 19A) and Log10 microneutralization (MN) titers (Figure 19B) against Sing16 virus are shown. [Figure 20] Figures 20A and 20B show T cell responses in NHPs vaccinated with Sing16 HA mRNA-LNP vaccine. Cynomolgus macaques (n=6 per group) were injected twice by IM route with 45, 135, or 250 μg of Sing16 HA mRNA-LNP, 4 weeks apart. T cells were assessed by ELISPOT on day 42 in PBMCs stimulated in vitro with a peptide pool representing the entire HA open reading frame. Responses of PBMCs secreting IFN-γ (Figure 20A) or IL-13 (Figure 20B), calculated as spot forming cells (SFC) per million PBMCs, are shown. Each symbol represents an individual sample and the bars represent the geometric mean for the group. [Figure 21]Figure 21 shows the secretion of Sing16 H3-specific IgG by memory B cells on day 180 in NHPs vaccinated with Sing16 HA mRNA-LNP vaccine. Cynomolgus macaques (n=6 per group) were injected twice by IM route with 15 or 45 μg Sing16 HA mRNA-LNP, 4 weeks apart. Sing16 / H3-specific and total IgG+ antibody-secreting cells (ASCs) were measured using a human IgG monochromatic memory B cell ELISPOT kit (CAT# NC1911372, CTL). Differentiation of MBCs into ASCs was performed in PBMCs collected on day 180 by using the stimulation cocktail provided by the kit. The number of IgG+ and Sing16 / H3-specific ASCs was calculated per million PBMCs for each animal, and the frequency of antigen-specific ASCs is shown. [Figure 22-1]Figure 22 shows the delivery of bivalent combinations of influenza vaccines in mice. BALB / c mice (n=8 per group) were immunized IM twice, 4 weeks apart, with 0.4 μg of bivalent combination co-encapsulated mRNA transcripts (1:1 wt / wt, half dose per leg) total or 0.2 μg of each monovalent separately formulated and immunized in different legs. CA09 HA mRNA-LNP, Sing16 HA mRNA-LNP constituting the H1H3 combo; Sing16 HA mRNA-LNP and Sing16 NA mRNA-LNP of the H3N2 combo and Mich15 NA mRNA-LNP and Perth09 NA mRNA-LNP of the N1N2 combo were tested against the corresponding viruses in serum collected on days 0, 14, 28, and 42. Panel (a) shows the HAI titers recorded against CA09 H1N1 influenza virus and Sing2016 H3N2. Panel (b) shows the HAI and NAI titers recorded for Sing2016 H3N2 and A / Mallard / Sweden / 2002(H6) chimeric influenza viruses and H6N2 A / Perth / 09 virus F1919D(N2) virus, respectively. Panel (c) shows the NAI titers recorded for Mich15(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus and H6N2 A / Perth / 09 virus F1919D(N2) virus. [Figure 22-2] Continued from Figure 22-1. [Figure 23]Figure 23 shows delivery of tetravalent combinations of influenza vaccines in NHPs. Cynomolgus macaques (n=6 per group) were immunized IM twice, 4 weeks apart, with 10 μg total of tetravalent combinations of co-encapsulated mRNA transcripts (1:1:1:1 wt / wt). H2H3N1N2 combo consisting of CA09 HA mRNA, Sing16 HA mRNA, Mich15 NA mRNA, and Perth09 NA mRNA. H1H3 combo consisting of CA09 HA mRNA, Sing16 HA mRNA, and 2× non-coding mRNA (ncmRNA); H3N2 combo of Sing16 HA mRNA and Perth09 NA mRNA and 2× non-coding mRNA. N1N2 combo of Mich15 NA mRNA, Perth09 NA mRNA-LNP, and 2× non-coding mRNA. H1 consisting of CA09 HA mRNA and 3× non-coding mRNA. H3 consisting of Sing16 HA mRNA and 3x non-coding mRNA. N1 consisting of Mich15 NA mRNA and 3x non-coding mRNA. N2 consisting of Perth09 NA mRNA and 3x non-coding mRNA. Inhibitory titers were tested against the corresponding viruses in serum collected on days 0, 14, 28, 42. Panel (a) shows the HAI titers recorded against CA09 H1N1 influenza virus and Sing16 H3N2. Panel (b) shows the NAI titers recorded against Mich15(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus and H6N2 Perth / 09 virus F1919D(N2) virus. [Figure 24] FIG. 24 depicts a graph showing the expression of human erythropoietin (hEPO) in mice treated with various LNP formulations of hEPO mRNA. LNP formulations "Lipid A", "Lipid B", "Lipid C", "Lipid D", and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Diagram 25]Figure 25 depicts a graph showing hEPO expression in non-human primates (NHPs) treated with various LNP formulations of hEPO mRNA. LNP formulations "Lipid A", "Lipid B", "Lipid C", "Lipid D", and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 26] FIG. 26 depicts a graph showing HAI titers at 28 and 42 days after injection of various LNP formulations of HA mRNA. LNP formulations "Lipid A", "Lipid B", "Lipid C", "Lipid D", and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 27] FIG. 27 depicts a graph showing Cal09 H1 HAI titers 28 and 42 days after injection of various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 28] FIG. 28 depicts a graph showing Sing16 H3 HAI titers at 28 and 42 days after injection of various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 29] FIG. 29 depicts the HAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to mice against four different influenza strains. [Diagram 30]FIG. 30 depicts the HINT values ​​for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. [Diagram 31] FIG. 31 depicts the NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to mice against four different influenza strains. [Diagram 32] Figure 32 depicts NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. Samples were obtained 20 days (D20) after the second dose of vaccine. [Diagram 33] Figure 33 depicts NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. Samples were obtained 42 days (D42) after the second dose of vaccine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The present disclosure provides a novel lipid nanoparticle (LNP) formulation for in vivo delivery of mRNA vaccines and a method for making the vaccine. The LNP is made of a mixture of four types of lipids: cationic lipids, polyethylene glycol (PEG)-conjugated lipids, cholesterol-based lipids, and helper lipids. The LNP encapsulates an mRNA molecule. The encapsulated mRNA molecule can contain naturally occurring ribonucleotides, chemically modified nucleotides, or a combination thereof, and can individually or collectively code for one or more proteins.

[0052] The present inventors discovered this formulation through screening a combinatorial library of lipid components. This LNP encapsulates and protects mRNA payload from degradation, and promotes cellular uptake of encapsulated mRNA. Compared to industrial formulations described in the literature, the LNP described herein enhances delivery efficiency and promotes endosomal escape of mRNA, as demonstrated by enhanced expression in vivo and in vitro, resulting in improved efficacy. For example, the LNPs disclosed herein have superior stability and / or efficacy profiles compared to known LNPs, such as heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (aka DLin-MC3-DMA or MC3; Semple et al., Nat Biotechnol. (2010) 28:172-6) or di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (aka L319; Maier et al., Mol Ther. (2013) 21(8):1570-8). As described further below, the present formulations encapsulating mRNA encoding hEPO, when delivered in vivo, resulted in high levels of erythropoietin circulating in the blood at 6 and 24 hours, up to a 12-fold increase compared to the industry standard, the MC3 LNP formulation. Similarly, high efficacy was found with other mRNAs, such as those encoding influenza antigens, in both mouse and non-human primate models.

[0053] The mRNA vaccines formulated herein can be used to induce a balanced immune response, including both cellular and humoral immunity. The advantage of the LNP formulations is that they are not sequence specific, so these formulations can be used to deliver mRNAs encoding various antigens, allowing for rapid deployment in epidemic or pandemic situations. Furthermore, the LNP-formulated mRNA vaccines are highly immunogenic, thus providing significant dose-sparing potential.

[0054] I. Lipid Nanoparticles (LNPs) The LNPs of the present disclosure include four categories of lipids: (i) ionizable lipids (eg, cationic lipids); (ii) pegylated lipids; (iii) cholesterol-based lipids, and (iv) helper lipids.

[0055] A. Ionizable lipids Ionizable lipids facilitate mRNA encapsulation and may be cationic lipids, which provide a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0056] In some embodiments, the cationic lipid is OF-02. [ka] OF-02 is a non-degradable structural analogue of OF-Deg-Lin. OF-Deg-Lin contains a degradable ester bond for binding to a diketopiperazine core and a double unsaturated tail, and OF-02 contains a non-degradable 1,2-amino alcohol bond for binding to the same diketopiperazine core and a double unsaturated tail (Fenton et al., Adv Mater. (2016) 28: 2939; U.S. Patent No. 10,201,618). The exemplary LNP formulation herein, lipid A, contains OF-2.

[0057] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Patent No. 9,512,073). [ka] In an exemplary LNP formulation herein, lipid B contains cKK-E10.

[0058] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1(2-(4-(2-((3-(bis((Z)-2-hydroxyoctadec-9-en-1-yl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate, which is a HEPES-based disulfide cationic lipid with a piperazine core and has the formula (III). [ka] An exemplary LNP formulation herein, lipid C, contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C has the same composition as lipid A or lipid B, except for the cationic lipid.

[0059] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((4-(bis(2-hydroxydecyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate, which is a HEPES-based disulfide cationic lipid with a piperazine core and has the formula (IV). [ka] An exemplary LNP formulation herein, lipid D, contains GL-HEPES-E3-E12-DS-4-E10. Lipid D has the same composition as lipid A or lipid B, except for the difference in the cationic lipid.

[0060] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate, which is a HEPES-based disulfide cationic lipid with a piperazine core and has the formula (V). [ka] An exemplary LNP formulation herein, Lipid E, contains GL-HEPES-E3-E12-DS-3-E14. Lipid E ​​has the same composition as Lipid A or Lipid B, except for the cationic lipid.

[0061] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1 (III), GL-HEPES-E3-E12-DS-4-E10 (IV), and GL-HEPES-E3-E12-DS-3-E14 (V) can be synthesized according to the general procedure presented in Scheme 1.

[0062] Scheme 1: General synthetic scheme for lipids of formulae (III), (IV), and (V) [ka]

[0063] In some embodiments, the cationic lipid is MC3 and has formula VI: [ka]

[0064] In some embodiments, the cationic lipid is SM-102 (9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid), having formula VII: [ka]

[0065] In some embodiments, the cationic lipid is ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoic acid), having formula VIII: [ka]

[0066] In some embodiments, the cationic lipid is [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoic acid (D-Lin-MC3-DMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); Di((Z)-non-2-en-l-yl) 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102);[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315);[3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-C hol);tetrakis(8-methylnonyl) 3,3',3",3'''-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl)tetrapropionate (306Oi10);decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9);ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B);1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexa (Octan-3-yl)9,9',9",9''',9"",9'''"-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9"Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoic acid) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid (Lipid 5); and combinations thereof.

[0067] In some embodiments, the cationic lipid is biodegradable.

[0068] In some embodiments, the cationic lipid is not biodegradable.

[0069] In some embodiments, the cationic lipid is cleavable.

[0070] In some embodiments, the cationic lipid is not cleavable.

[0071] Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60 2014); Fenton et al. (Adv Mater. 28:2939 2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference.

[0072] B. PEGylated lipids The PEGylated lipid moiety controls the particle size and stability of the nanoparticles. The addition of such moieties can provide a means to prevent complex aggregation and increase circulation life and delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters 268(1):235-7, 1990). These moieties can be selected to rapidly clear from the pharmaceutical composition in vivo (see, e.g., U.S. Pat. No. 5,885,613).

[0073] Contemplated PEGylated lipids include C6-C ceramides, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (For example, C8, C 10 , C 12 , C 14 , C 16 , or C 18PEGylated lipids include, but are not limited to, polyethylene glycols (PEGs) up to 5 kDa in length covalently attached to lipids having an alkyl chain(s) of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropyl carbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0074] In some embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the pegylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the pegylated lipid herein is DMG-PEG2000.

[0075] C. Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res. Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol esters ("ICE"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanostadiene (Δ5,7-cholesterol); LNPs include, but are not limited to, 24,25-dihydrolanosterol; zymosterol (5α-cholest-8,24-dien-3β-ol); lathosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campestrol (campest-5-en-3β-ol); campestanol (5α-campestan-3b-ol); 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarete (cholest-5-en-3β-ylheptadecanoic acid); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNPs is cholesterol.

[0076] D. Helper lipids The helper lipid enhances the structural stability of the LNP and aids in the endosomal escape of the LNP. The helper lipid improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid, which has membrane fusogenic properties to enhance the uptake and release of the drug payload. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0077] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0078] In various embodiments, the LNP comprises (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0079] E. Molar ratio of lipid components The molar ratio of the above components is important for the effectiveness of the LNP in delivering mRNA. The molar ratio of cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid is A:B:C:D, where A+B+C+D=100%. In some embodiments, the molar ratio of cationic lipid in the LNP compared to total lipid (i.e., A) is 35-55%, such as 35-50% (e.g., 38-42%, such as 40%, or 45-50%). In some embodiments, the molar ratio of the PEGylated lipid component compared to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, such as 1.5%). In some embodiments, the molar ratio of the cholesterol-based lipid compared to total lipid (i.e., C) is 20-50% (e.g., 27-30%, such as 28.5%, or 38-43%). In some embodiments, the molar ratio of helper lipid relative to total lipid (i.e., D) is 5-35% (e.g., 28-32%, such as 30%, or 8-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of cationic lipid to helper lipid that is greater than 1.

[0080] In certain embodiments, the LNPs of the disclosure include cationic lipid at a molar ratio of 35%-55% or 40%-50% (e.g., cationic lipid at a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%); polyethylene glycol (PEG)-conjugated (pegylated) lipids at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., pegylated lipids at a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); Cholesterol-based lipids at a molar ratio of 20%-50%, 25%-45%, or 28.5%-43% (e.g., cholesterol-based lipids at a molar ratio of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%); and Helper lipids at molar ratios of 5%-35%, 8%-30%, or 10-30% (e.g., helper lipids at molar ratios of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%) Including, All molar ratios are relative to the total lipid content of the LNP.

[0081] In certain embodiments, the LNPs comprise a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and Helper lipid is included at a molar ratio of 30%.

[0082] In certain embodiments, the pegylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0083] In various embodiments, the cholesterol-based lipid is cholesterol.

[0084] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0085] In certain embodiments, the LNPs comprise OF-02 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0086] In certain embodiments, the LNPs comprise cKK-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0087] In certain embodiments, the LNPs comprise GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0088] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0089] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0090] In certain embodiments, the LNPs comprise SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0091] In certain embodiments, the LNPs comprise ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0092] In certain embodiments, the LNP comprises OF-02 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid A".

[0093] In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid B".

[0094] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid C".

[0095] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid D".

[0096] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid E".

[0097] In certain embodiments, the LNPs comprise 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid (SM-102) at a molar ratio of 50%, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%, cholesterol at a molar ratio of 38.5%; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0098] In certain embodiments, the LNPs comprise (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoic acid) (ALC-0315) in a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 9.4%; cholesterol in a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.6%.

[0099] In certain embodiments, the LNPs comprise [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoic acid) (ALC-0315) at a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%, cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.7%.

[0100] To calculate the actual amount of each lipid in the LNP formulation, the molar amount of the cationic lipid is first determined based on the desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP. The molar amount of each of the other lipids is then calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weights using the molecular weight of each lipid.

[0101] F. Active ingredient of LNP The active ingredient of the present LNP vaccine composition is the mRNA that encodes the influenza antigen.

[0102] If desired, the LNPs may be multivalent. In some embodiments, the LNPs may carry mRNAs encoding more than one influenza antigen, such as two, three, four, five, six, seven, or eight influenza antigens. For example, the LNPs may carry multiple mRNAs, each encoding a different influenza antigen; or carry polycistronic mRNAs (e.g., each antigen coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide, such as the 2A peptide) that can be translated into more than one influenza antigen. LNPs carrying different mRNAs typically contain (encapsulate) multiple copies of each mRNA. For example, LNPs carrying or encapsulating two different mRNAs typically carry multiple copies of each of the two different mRNAs.

[0103] In some embodiments, a single LNP formulation may contain multiple types of LNPs (e.g., two, three, four, five, six, seven, eight, nine, ten, or more), with each type carrying a different mRNA.

[0104] In some embodiments, the multivalent LNP vaccine contains mRNA molecules encoding polypeptides derived from eight influenza virus proteins selected from hemagglutinin (e.g., hemagglutinin 1 (HA1) and hemagglutinin 2 (HA2)), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), and nonstructural protein 2 (NS2). In further embodiments, the multivalent LNP vaccine contains eight mRNAs encoding antigenic polypeptides derived from the HA protein, from the NA protein, and from both the HA and NA proteins. In some embodiments, the mRNAs encoding the antigenic polypeptides are from different influenza strains.

[0105] In certain embodiments, the composition may comprise one or more mRNAs encoding antigens of influenza A, B and C viruses. In one embodiment, the composition may comprise one or more mRNAs encoding HA and / or NA antigens of influenza A and influenza B viruses. In one embodiment, the HA antigen of the influenza A virus is selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. In one embodiment, the NA antigen of the influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. In one embodiment, the HA and NA antigens of the influenza B virus are from the influenza B / Yamagata lineage. In one embodiment, the HA and NA antigens of the influenza B virus are from the influenza B / Victoria lineage. In some embodiments, one or more of the HA and NA antigens are derived from an influenza virus strain recommended by the World Health Organization (WHO) in its annual recommendations for influenza vaccine formulations.

[0106] In certain embodiments, at least one of the one or more influenza virus proteins comprises an influenza virus HA protein and / or an influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and at least one of the one or more ribonucleic acid molecules encodes one or more influenza virus proteins having a molecular sequence identified or designed from a machine learning model.

[0107] In one embodiment, the composition comprises one mRNA encoding an H3 HA antigen, one mRNA encoding an H1 HA antigen, one mRNA encoding an HA antigen from the influenza B / Yamagata lineage, and one mRNA encoding an HA antigen from influenza B / Victoria.

[0108] In one embodiment, the composition comprises one mRNA encoding an H3 HA antigen, one mRNA encoding an N2 NA antigen, one mRNA encoding an H1 HA antigen, one mRNA encoding an N1 NA antigen, one mRNA encoding an HA antigen from influenza B / Yamagata lineage, one mRNA encoding an NA antigen from influenza B / Yamagata lineage, one mRNA encoding an HA antigen from influenza B / Victoria lineage, and one mRNA encoding an NA antigen from influenza B / Victoria lineage.

[0109] In embodiments, the composition further comprises one or more mRNAs encoding a machine-learned influenza virus HA having a molecular sequence identified or designed from a machine-learning model, wherein the one or more machine-learned influenza virus HAs may be selected from an H1 HA, an H3 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, or a combination thereof.

[0110] When selecting one or more machine learning influenza virus HAs, any machine learning algorithm may be used, for example, any of the machine learning algorithms and methods disclosed in PCT Publication No. WO 2021 / 080990, entitled SYSTEMS AND METHODS FOR DESIGNING VACCINES, and PCT Publication No. WO 2021 / 080999, entitled SYSTEMS AND METHODS FOR PREDICTING BIOLOGICAL RESPONSES, both of which are incorporated herein by reference in their entireties.

[0111] The mRNA may be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or chemically modified (e.g., containing nucleotide analogs such as pseudouridines (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate linkages). The mRNA molecule may include a 5' cap and a poly-A tail.

[0112] G. Buffers and other components To stabilize the nucleic acid and / or LNP (e.g., to extend the shelf life of a vaccine product), to facilitate administration of the LNP pharmaceutical composition, and / or to enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharma- ceutically acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0113] The LNP compositions of the present disclosure can be provided in frozen liquid form or in lyophilized form. A variety of cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition includes trehalose, for example, at 5-30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP composition can be frozen (or lyophilized and stored frozen) at -20°C to -80°C.

[0114] The LNP composition may be given to the patient in an aqueous buffer solution, if previously frozen or previously lyophilized, thawed and reconstituted with an aqueous buffer solution at bedside. The buffer solution is preferably isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0115] II. RNA The LNP vaccine compositions of the present disclosure may include an RNA molecule (e.g., mRNA) encoding an antigen of interest. The RNA molecule of the present disclosure may include at least one ribonucleic acid (RNA) that includes an ORF encoding an antigen of interest. In certain embodiments, the RNA is a messenger RNA (mRNA) that includes an ORF encoding an antigen of interest. In certain embodiments, the RNA (e.g., mRNA) further includes at least one 5'UTR, 3'UTR, poly(A) tail, and / or 5' cap.

[0116] II.A.5' Cap The mRNA 5' cap confers resistance to nucleases found in most eukaryotic cells and can enhance translation efficiency. Several types of 5' caps are known: 7-methylguanosine cap ("m 7 The cap sequence (also referred to as "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide through a 5'-5' triphosphate bond.

[0117] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase, generating a 5'5'5 triphosphate linkage; and then the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication No. 2016 / 0032356 and U.S. Patent Application Publication No. 2018 / 0125989, which are incorporated herein by reference.

[0118] 5'-capping of polynucleotides may be completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies), which yield a 5'-guanosine cap structure according to the manufacturer's protocol. 5'-capping of modified RNA may be completed post-transcriptionally using vaccinia virus capping enzyme to generate the cap 0 structure: m7G(5')ppp(5')G. Cap 1 structure may be generated using both vaccinia virus capping enzyme and 2'-O methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from cap 1 structure, followed by 2'-O methylation of the 5'-antepenultimate nucleotide using 2'-O methyltransferase. Cap 3 structure may be generated from cap 2 structure, followed by 2'-O methylation of the 5'-preantepenultimate nucleotide using 2'-O methyltransferase.

[0119] In certain embodiments, an mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0120] In certain embodiments, the mRNA of the present disclosure comprises: [ka] Contains the 5' cap.

[0121] II.B. Untranslated Regions (UTRs) In some embodiments, the mRNA of the present disclosure comprises a 5' and / or 3' untranslated region (UTR). In the mRNA, the 5'UTR starts at the transcription initiation site and continues up to but not including the start codon. The 3'UTR starts immediately following the stop codon and continues to the transcription termination signal.

[0122] In some embodiments, the mRNA disclosed herein may comprise a 5'UTR that includes one or more elements that affect mRNA stability or translation. In some embodiments, the 5'UTR may be about 10-5,000 nucleotides in length. In some embodiments, the 5'UTR may be about 50-500 nucleotides in length. In some embodiments, the 5'UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 65 nucleotides in length, or about 70 nucleotides in length. 0 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or 5,000 nucleotides in length.

[0123] In some embodiments, the mRNAs disclosed herein may include a 3'UTR that includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in a cell, or one or more binding sites for an miRNA. In some embodiments, the 3'UTR may be about 50-5,000 nucleotides in length or longer. In some embodiments, the 3'UTR may be about 50-1,000 nucleotides in length or longer. In some embodiments, the 3'UTR is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides long.

[0124] In some embodiments, the mRNAs disclosed herein may include a 5' or 3' UTR that is derived from a gene different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0125] In certain embodiments, the 5' and / or 3' UTR sequences can be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, the 5' UTR sequence may include a partial sequence of the CMV immediate early 1 (IE1) gene, or a fragment thereof, to improve nuclease resistance and / or improve the half-life of the mRNA. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof in the 3' end or untranslated region of the mRNA. In general, these modifications include modifications made to improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA compared to its unmodified counterpart, e.g., to improve such mRNA resistance to in vivo nuclease digestion.

[0126] Exemplary 5'UTRs include sequences from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication No. 2014 / 0206753 and U.S. Patent Application Publication No. 2015 / 0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO:22) (U.S. Patent Application Publication No. 2016 / 0151409, which is incorporated herein by reference).

[0127] In various embodiments, the 5'UTR may be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication No. 2017 / 0029847, U.S. Patent Application Publication No. 2016 / 0304883, U.S. Patent Application Publication No. 2016 / 0235864, and U.S. Patent Application Publication No. 2016 / 0166710, each of which is incorporated herein by reference).

[0128] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, see above).

[0129] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, see above).

[0130] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, see above). In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0131] In some embodiments, the 5'UTR comprises the nucleic acid sequence shown in SEQ ID NO:19 and reproduced below. GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 19)

[0132] In some embodiments, the 3'UTR comprises the nucleic acid sequence shown in SEQ ID NO:20 and reproduced below. CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 20)

[0133] The 5'UTR and 3'UTR are described in further detail in WO2012 / 075040, which is incorporated herein by reference.

[0134] II.C. Polyadenylation Tail As used herein, the terms "poly(A) sequence", "poly(A) tail" and "poly(A) region" refer to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. The poly(A) tail may provide stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have a length of essentially 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide that is different from adenosine nucleotides (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide that is different from adenosine nucleotides, or a stretch of nucleotides). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 23).

[0135] A "poly(A) tail", as used herein, typically relates to RNA. However, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0136] The poly(A) tail may comprise from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0137] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription.In certain embodiments, the poly(A) tail is obtained by the general method of chemical synthesis without being transcribed from a DNA template.In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of RNA using a commercially available polyadenylation kit and corresponding protocol, or alternatively, by using the method and means described in WO2016 / 174271, using immobilized poly(A) polymerase.

[0138] The nucleic acid may comprise a poly(A) tail resulting from enzymatic polyadenylation, with the majority of nucleic acid molecules comprising from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0139] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from the template DNA and may further comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO2016 / 091391.

[0140] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0141] In various embodiments, the nucleic acid may include at least one poly(C) sequence.

[0142] The term "poly(C) sequence," as used herein, is intended to mean a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0143] II.D. Chemical modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may include at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNAs may be any of the following: 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo ... The nucleic acids may be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines such as mo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioates, peptide nucleotides, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0144] In some embodiments, the disclosed mRNAs may contain at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0145] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0146] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0147] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0148] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0149] The preparation of such analogs is described, for example, in U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642.

[0150] II.E. mRNA synthesis The mRNA disclosed herein may be synthesized according to any of a wide variety of methods. For example, the mRNA according to the present disclosure may be synthesized by in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al., (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al., (2013) Methods Enzymol. 530: 101-14. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions may vary according to the particular application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. Although mRNA provided from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0151] III. Methods for Making the Present LNP Vaccines The LNPs can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques such as dissolving lipids in a suitable solvent and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying to deposit the selected lipids on the inner wall of a suitable container or vessel. An aqueous phase can then be added to the vortex vessel to form MLVs. Unilamellar liposomes (ULVs) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. Additionally, unilamellar liposomes can be formed by detergent removal techniques.

[0152] Various methods are described in US2011 / 0244026, US2016 / 0038432, US2018 / 0153822, US2018 / 0125989, and PCT / US2020 / 043223 (filed July 23, 2020) and can be used to carry out the present invention. One exemplary method involves encapsulating the mRNA by mixing it with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in US2016 / 0038432. Another exemplary method involves encapsulating the mRNA by mixing preformed LNPs with the mRNA, as described in US2018 / 0153822.

[0153] In some embodiments, the method of making mRNA-loaded LNPs comprises heating one or more of the solutions to a temperature higher than the ambient temperature, the one or more solutions being a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing LNP-encapsulated mRNA. In some embodiments, the method comprises heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step. In some embodiments, the method comprises heating one or more of the solution containing preformed LNPs, the solution containing mRNA, and the solution containing LNP-encapsulated mRNA during the mixing step. In some embodiments, the method comprises heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions are heated is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature to which one or more of the solutions are heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature is about 65° C.

[0154] A variety of methods may be used to prepare mRNA solutions suitable for the present invention. In some embodiments, the mRNA may be dissolved directly in a buffer solution as described herein. In some embodiments, the mRNA solution may be made by mixing the mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, the mRNA solution may be made by mixing the mRNA stock solution with a buffer solution just before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or higher in water or buffer.

[0155] In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than the rate of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min, or greater.

[0156] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or greater.

[0157] The method of incorporating desired mRNA into lipid nanoparticles is called "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. LNP-incorporated nucleic acid may be located completely or partially in the internal space of lipid nanoparticles, within the bilayer membrane of lipid nanoparticles, or associated with the outer surface of lipid nanoparticle membrane. Incorporation of mRNA into lipid nanoparticles is also referred to herein as "encapsulation", where the nucleic acid is completely or substantially contained within the internal space of lipid nanoparticles.

[0158] Suitable LNP can be made in various sizes.In some embodiments, the reduction in size of lipid nanoparticles is associated with more efficient delivery of mRNA.Selection of suitable LNP size may take into account the site of target cell or tissue and to some extent the application for which lipid nanoparticles are made.

[0159] A wide variety of methods known in the art are available for sizing a population of lipid nanoparticles. A preferred method herein utilizes a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed in the Zetasizer machine. The z-average diameter (nm), or cumulant average, is considered as the average size for the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) evaluation to guide efficient lipid nanoparticle synthesis.

[0160] In some embodiments, the majority of the purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0161] In some embodiments, the LNPs in the composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0162] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LNPs in the composition have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm), or about 50-70 nm (e.g., 55-65 nm), which are particularly suitable for pulmonary delivery via inhalation administration.

[0163] In some embodiments, the dispersity, or molecular size heterogeneity measure (PDI), of the LNPs in the pharmaceutical compositions provided herein is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI may be measured by a Zetasizer machine as described above.

[0164] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein have mRNA encapsulated within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition have mRNA encapsulated within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 90%, or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).

[0165] In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio of above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0166] In some embodiments, pharmaceutical compositions according to the invention contain at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, pharmaceutical compositions contain at least about 0.1 μg-1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA.

[0167] In some embodiments, mRNA can be produced by chemical synthesis or by in vitro transcription (IVT) of DNA template. An exemplary method for producing and purifying mRNA is described in Example 1. In this method, in the IVT method, a cDNA template is used to produce mRNA transcripts, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is purified again by depth filtration and TFF.

[0168] The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP may be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer may contain other components such as salts (e.g., sodium, potassium, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, pH 4.5.

[0169] An exemplary non-limiting method for making mRNA-LNP compositions is described in Example 1. The method involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled and homogenous manner, with the lipid to mRNA ratio being maintained throughout the mixing method. In this example, the mRNA is presented in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed in a 4:1 volume ratio in a "T" mixer with an approximately "pulseless" pump system. The resulting mixture then undergoes downstream purification and buffer exchange. Buffer exchange may be achieved using a dialysis cassette or a TFF system. TFF may be used to concentrate and buffer exchange the nascent LNPs obtained immediately after formation by the T-mixing method. The diafiltration process is a continuous operation and the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow.

[0170] IV. Packaging and Use of mRNA-LNP Vaccines The mRNA-LNP vaccine can be packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or nasopharyngeal (e.g., intranasal) administration. The vaccine composition can be in the form of an extemporaneous formulation, where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately prior to use. The vaccine composition can be shipped and provided in the form of an aqueous or frozen aqueous solution, and can be administered directly to a subject without reconstitution (after thawing if previously frozen).

[0171] Thus, the present disclosure provides a kit-like product that provides the mRNA-LNP vaccine in a single container, or provides the mRNA-LNP vaccine in one container and a physiological buffer for reconstitution in another container. The container(s) may contain single-use or multi-use dosages. The container may be a pre-processed glass vial or ampoule. The product may also include instructions for use.

[0172] In certain embodiments, the mRNA-LNP vaccine is provided for use in intramuscular (IM) injection. The vaccine can be injected into the deltoid muscle of the upper arm of the subject, for example. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for use in inhalation, and is provided in a pre-filled pump, intratracheal nebulizer spray, or inhaler.

[0173] The mRNA-LNP vaccine can be administered to a subject in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against the target pathogen for a sufficient time (e.g., 1 year, 2 years, 5 years, 10 years, or lifelong). Sufficient immune protection can be, for example, prevention or reduction of symptoms associated with infection by the pathogen. In some embodiments, multiple doses (e.g., two doses) of the vaccine are injected into a subject in need thereof to achieve a desired prophylactic effect. The administrations (e.g., prime and boost administrations) can be separated by intervals of, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, or 10 years.

[0174] In some embodiments, a single dose of mRNA-LNP vaccine contains 1-50 μg of mRNA (e.g., monovalent or multivalent). For example, a single dose may contain about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg of mRNA for intramuscular (IM) injection. In further embodiments, a multivalent single dose LNP vaccine contains multiple (e.g., 2, 3, or 4) types of LNPs, each for a different antigen, with each type of LNP having, for example, an mRNA amount of about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg.

[0175] In another aspect, the present invention provides a method for immunizing a subject against one or more influenza viruses in a subject. The present invention further provides a method for inducing an immune response against one or more influenza viruses in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a composition described herein.

[0176] In various embodiments, the immunization methods provided herein induce a broad protective immune response against multiple epitopes within one or more influenza viruses. In various embodiments, the immunization methods provided herein induce a broad neutralizing immune response against one or more influenza viruses. In some embodiments, the immune response comprises an antibody response. Thus, in various embodiments, the compositions described herein can provide broad cross-protection against different types of influenza viruses. In some embodiments, the compositions provide cross-protection against avian, swine, seasonal, and / or pandemic influenza viruses. In some embodiments, the compositions provide cross-protection against one or more influenza A, B, or C subtypes. In some embodiments, the compositions provide cross-protection against multiple strains of influenza A H1 subtype viruses (e.g., H1N1), influenza A H3 subtype viruses (e.g., H3N2), influenza A H5 subtype viruses (e.g., H5N1), and / or influenza B viruses (e.g., Yamagata lineage, Victoria lineage).

[0177] In some embodiments, the methods of the present invention can elicit an improved immune response against one or more seasonal influenza strains. Exemplary seasonal strains include A / Puerto Rico / 8 / 1934, A / Fort Monmouth / 1 / 1947, A / Chile / 1 / 1983, A / Texas / 36 / 1991, A / Singapore / 6 / 1986, A / Beijing / 32 / 1992, A / New Caledonia / 20 / 1999, A / Solomon A / Brisbane / 03 / 2006, A / Brisbane / 59 / 2007, A(H3N2) viruses antigenically similar to the cell-transmitted prototype virus A / Victoria / 361 / 2011, A / Beijing / 262 / 95(H1N1)-like viruses, A / Brisbane / 02 / 2018(H1N1)pdm09-like viruses, A / Brisbane / 10 / 2007(H3N2)-like viruses, A / California / 7 / 2004(H3N2)-like viruses, A / California / 7 / 2009(H1 N1)-like viruses, A / California / 7 / 2009(H1N1)pdm09-like viruses, A / Cambodia / e0826360 / 2020(H3N2)-like viruses, A / Fujian / 411 / 2002(H3N2)-like viruses, A / Fujian / 411 / 2002(H3N2)-like viruses, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09-like viruses, A / Hawaii / 70 / 2019(H1N1)pdm09-like viruses, A / Hong Kong / Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Hong Kong / 4801 / 2014(H3N2)-like virus, A / Kansas / 14 / 2017(H3N2)-like virus, A / Michigan / 45 / 2015(H1N1)pdm09-like virus, A / Moscow / 10 / 99(H3N2)-like virus, A / New Caledonia / 20 / 99(H1N1)-like virus, A / Perth / 16 / 2009(H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2)-like virus, A / SolomonIslands / 3 / 2006(H1N1)-like virus, A / South Australia / 34 / 2019(H3N2)-like virus, A / Switzerland / 8060 / 2017(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / Sydney / 5 / 97(H3N2)-like virus, A / Texas / 50 / 2012(H3N2)-like virus, A / Victoria / 2570 / 2019(H1N1)pdm09-like virus, A / Victoria / 361 / 2011(H3N2)-like virus , A / Wellington / 1 / 2004(H3N2)-like viruses, A / Wisconsin / 588 / 2019(H1N1)pdm09-like viruses, A / Wisconsin / 588 / 2019(H1N1)pdm09-like viruses, A / Wisconsin / 67 / 2005(H3N2)-like viruses, B / Beijing / 184 / 93-like viruses, B / Brisbane / 60 / 2008-like viruses, B / Colorado / 06 / 2017-like viruses (B / Victoria / 2 / 87 lineage), B / Florida / 4 / 2006-like viruses, B / Hong Kong ...including, but not limited to, Kong / 330 / 2001-like viruses, B / Malaysia / 2506 / 2004-like viruses, B / Massachusetts / 2 / 2012-like viruses, B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like viruses, B / Phuket / 3073 / 2013-like viruses, B / Phuket / 3073 / 2013-like viruses (B / Yamagata / 16 / 88 lineage), B / Shangdong / 7 / 97-like viruses, B / Shanghai / 361 / 2002-like viruses, B / Sichuan / 379 / 99-like viruses, B / Washington / 02 / 2019 (B / Victoria lineage)-like viruses, B / Washington / 02 / 2019-like (B / Victoria lineage) viruses, and B / Wisconsin / 1 / 2010-like viruses. In some embodiments, the methods of the present invention can induce an improved immune response against one or more pandemic influenza strains. Exemplary pandemic strains include, but are not limited to, A / California / 07 / 2009, A / California / 04 / 2009, A / Belgium / 145 / 2009, A / South Carolina / 01 / 1918, and A / New Jersey / 1976. Pandemic subtypes include, among others, H1N1, H5N1, H2N2, H3N2, H9N2, H7N7, H7N3, H7N9, and H10N7 subtypes. In some embodiments, the methods of the present invention can induce an improved immune response against one or more swine influenza strains. Exemplary swine strains include, but are not limited to, A / New Jersey / 1976 isolates and A / California / 07 / 2009. In some embodiments, the methods of the present invention can induce an improved immune response against one or more avian influenza strains. Exemplary avian strains include, but are not limited to, H5N1, H7N3, H7N7, H7N9, and H9N2. Additional influenza pandemic, seasonal, avian and / or swine strains are known in the art.

[0178] In some embodiments, the present invention provides a method of preventing or treating influenza infection by administering a composition of the present invention to a subject in need thereof. In some embodiments, the subject has or is susceptible to influenza infection. In some embodiments, the subject is considered to have influenza infection if the subject exhibits one or more symptoms commonly associated with influenza infection. In some embodiments, the subject is known or believed to have been exposed to influenza virus. In some embodiments, the subject is considered to be susceptible to influenza infection if the subject is known or believed to have been exposed to influenza virus. In some embodiments, the subject is known or believed to have been exposed to influenza virus if the subject is in contact with other individuals known or suspected to be infected with influenza virus and / or the subject is in or has been in a location where influenza infection is known or believed to be prevalent.

[0179] In various embodiments, the compositions described herein may be administered prior to or after the onset of one or more symptoms of influenza infection. In some embodiments, the compositions are administered as prophylactic agents. In such embodiments, the methods of the invention are effective in preventing or protecting a subject from influenza virus infection. In some embodiments, the compositions of the invention are used as a component of seasonal and / or pandemic influenza vaccines or as part of an influenza vaccination regimen intended to provide long-lasting (multi-season) protection. In some embodiments, the compositions of the invention are used to treat symptoms of influenza infection.

[0180] In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a livestock or pet (e.g., dog, cat, sheep, cow, and / or pig). In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a bird (e.g., chicken).

[0181] In some embodiments, the subject is a human. In certain embodiments, the subject is an adult, an adolescent, or an infant. In some embodiments, the human subject is younger than 6 months of age. In some embodiments, the human subject is 6 months or older, 6 months to 35 months, 36 months to 8 years, or 9 years or older. In some embodiments, the human subject is 60 years or older, or 55 years or older, such as 65 years or older. Administration of the compositions and / or carrying out the treatment methods in utero is also contemplated hereby.

[0182] Unless otherwise defined herein, scientific and technical terms used in connection with this specification shall have the same meaning as commonly understood by those skilled in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein may also be used in practicing or testing the present invention. In case of conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the universal and general knowledge in the art. As used herein, the terms "approximately" or "about" applied to one or more values ​​of interest refer to values ​​that are similar to the stated reference value. In certain embodiments, the term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (higher or lower) of a stated reference value, unless otherwise stated or apparent from the context.

[0183] V. Vector In one aspect, a vector comprising the mRNA composition disclosed herein is disclosed herein. The RNA sequence encoding the protein of interest (e.g., the mRNA encoding an influenza protein) can be cloned into several types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0184] In certain embodiments, vectors can be used to express mRNA in host cells. In various embodiments, vectors can be used as templates for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0185] In some embodiments, the vectors disclosed herein can include at least the following, from 5' to 3': an RNA polymerase promoter, a polynucleotide sequence encoding a 5'UTR, a polynucleotide sequence encoding an ORF, a polynucleotide sequence encoding a 3'UTR, and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein can include a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0186] A variety of RNA polymerase promoters are known. In some embodiments, the promoter can be a T7 RNA polymerase promoter. Other useful promoters can include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences for T7, T3 and SP6 promoters are known.

[0187] Also disclosed herein are host cells (eg, mammalian cells, eg, human cells) that contain the vectors or RNA compositions disclosed herein.

[0188] Polynucleotides can be introduced into target cells using any of several different methods, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa et al. (2001). Hum Gene Ther. 12(8):861-70), or the TransIT-RNA transfection kit (Mirus, Madison, Wis.).

[0189] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0190] Regardless of the method used to introduce exogenous nucleic acid into a host cell, or alternatively to expose the cell to an inhibitor of the present disclosure, a variety of assays may be performed to confirm the presence of the mRNA sequence in the host cell.

[0191] VI. Self-replicating and trans-replicating RNAs self-replicating RNA In one aspect, disclosed herein is a self-replicating RNA that encodes an influenza protein.

[0192] Self-replicating RNAs can be produced, for example, by using replication elements from alphaviruses and replacing structural viral proteins with nucleotide sequences that code for a protein of interest (e.g., influenza proteins). Self-replicating RNAs are typically positive-stranded molecules that can be translated immediately after delivery to a cell, providing an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA produces multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may themselves be translated to result in the in situ expression of the encoded antigen (i.e., influenza protein antigen) or may be transcribed to result in further transcripts with the same sense as the delivered RNA that are translated to result in the in situ expression of the antigen. The overall result of this sequence of transcription is a large amplification of the number of introduced replicon RNAs, such that the encoded antigen becomes the major polypeptide product of the cell.

[0193] One suitable system for achieving self-replication in this manner is to use alphavirus-based replicons. These replicons are positive-sense RNAs that lead to the translation of replicase (or replicase-transcriptase) after delivery to the cell. The replicase is translated as a polyprotein to provide a replication complex that self-cleaves to create a genomic strand copy of the positive-sense delivered RNA. These negative-sense transcripts are themselves transcribed to provide further copies of the positive-sense parent RNA, as well as subgenomic transcripts that code for antigens. Translation of the subgenomic transcripts thus leads to in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can be used from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used, for example, the attenuated TC83 mutant of VEEV has been used in replicons. See the following reference: WO2005 / 113782, which is incorporated herein by reference.

[0194] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) an influenza protein antigen. The polymerase can be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Although naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, the self-replicating RNA can produce its own genomic RNA copy in the cell, but cannot produce RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot persist in an infectious form. The alphavirus structural proteins necessary for wild-type virus persistence are absent from the self-replicating RNA of the present disclosure, and their place is taken by a gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes an immunogen rather than a structural alphavirus virion protein. Self-replicating RNA is described in further detail in WO2011005799, which is incorporated herein by reference.

[0195] Trans-replicating RNA In one aspect, disclosed herein is a trans-replicating RNA that encodes an influenza protein.

[0196] Trans-replicating RNA has elements similar to the self-replicating RNA described above. However, in the case of trans-replicating RNA, two separate RNA molecules are used. The first RNA molecule encodes the above-mentioned RNA replicase (e.g., alphavirus replicase), and the second RNA molecule encodes a protein of interest (e.g., influenza protein antigen). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby greatly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNA is described in more detail in WO2017162265, which is incorporated herein by reference.

[0197] VII. Pharmaceutical Compositions RNA purified according to the present disclosure can be useful as a component in pharmaceutical compositions for use, for example, as a vaccine. These compositions typically include RNA and a pharma- ceutically acceptable carrier. The pharmaceutical compositions of the present disclosure can also include one or more additional components, such as a small molecule immunostimulant (e.g., a TLR agonist). The pharmaceutical compositions of the present disclosure can also include a delivery system for RNA, such as a liposome, an oil-in-water emulsion, or a microparticle. In some embodiments, the pharmaceutical composition includes a lipid nanoparticle (LNP). In certain embodiments, the composition includes an antigen-encoding nucleic acid molecule encapsulated within the LNP.

[0198] VIII. Vaccination Methods The influenza vaccines disclosed herein may be administered to a subject to induce an immune response directed to one or more influenza proteins, and the anti-antigen antibody titer in the subject is increased following vaccination compared to the anti-antigen antibody titer in a subject not vaccinated with the influenza vaccine disclosed herein, or compared to another vaccine against influenza. An "anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0199] In one aspect, the present disclosure provides a method of inducing an immune response against influenza or protecting a subject from influenza infection, comprising administering to a subject an influenza vaccine as described herein.The present disclosure also provides an influenza vaccine as described herein for use in inducing an immune response against influenza or protecting a subject from influenza infection.The present disclosure also provides an influenza mRNA as described herein for use in the manufacture of a vaccine for inducing an immune response against influenza or protecting a subject from influenza infection.

[0200] In order that this invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. EXAMPLES

[0201] Optimization of LNP formulation This example describes a series of LNP formulations for mRNA vaccines produced from combinatorial libraries of various components. A new rationally designed cationic lipid was synthesized. In total, more than 150 lipids and more than 430 formulations were tested. Human erythropoietin (hEPO) mRNA was used as the test mRNA. In the main formulation described below, mRNA is formulated into LNP using cationic lipids and combinations of three other lipids: helper lipids; cholesterol-based lipids; and PEGylated lipids, in various permutations.

[0202] The LNP formulation consists of four lipid components: ionizable lipid, helper lipid DOPE, cholesterol, and PEGylated lipid DMG-PEG-2K. The PEGylated lipid molar fraction was kept constant at 1.5%, and the ionizable lipid and different helper lipids and their molar ratios were evaluated to identify optimized ratios based on hEPO screening studies.

[0203] Citrate buffer (1 mM citric acid, 150 mM NaCl, pH 4.5) was used to prepare the LNP formulation. The mRNA solution added to the citrate buffer was mixed with lipids in ethanol solution during the formulation process. The pH and concentration of the buffer were selected to achieve a high rate of mRNA encapsulation in the LNP formulation.

[0204] The LNP formulation method involved mixing the lipid ethanol solution and the mRNA citrate solution in a "T" mixer using a pump system. The resulting solution then underwent buffer exchange using TFF / dialysis tubing. The concentration of the final formulation in 10% (w / v) trehalose was adjusted based on dosing requirements.

[0205] Mouse in vivo expression of hEPO protein was used as a surrogate to measure the efficacy of LNPs to deliver mRNA in vivo. In this study, a single dose (0.1 μg) of hEPO mRNA formulated in LNPs from various combinations of components was injected intramuscularly (IM) into mice. Serum collected 6 and 24 hours after administration was tested for hEPO levels using ELISA. The industry benchmark MC3 formulation was used as a reference for calculation of fold increase in hEPO expression (Angew, Chem Int Ed. (2012) 51:8529-33).

[0206] The level of hEPO expression observed for each LNP formulation indicated the formulation's ability to deliver mRNA into cells. The first formulation contained 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; helper lipid), DMG-PEG2000, and cholesterol in the molar ratio of cationic lipid:DMG-PEG2000:cholesterol:DOPE of 40:1.5:28.5:30. These formulations were found to have robust efficacy compared to the MC3 formulation.

[0207] Further formulations were tested. The optimized formulations Lipid A LNP and Lipid B LNP are shown in Table 1. The mRNA in these formulations can be modified or unmodified and can code for influenza-derived antigens.

[0208] [Table 1] In Table 1, the final dose for human vaccine would be a dilution of the above final bulk product in phosphate buffered saline (PBS) based on the intended single human dose. The WFI amount is calculated based on the nominal final formulation. The trehalose content in the formulation is equivalent to 10% (100 mg / mL) trehalose dihydrate, converted to the anhydrous component using the ratio of the molecular weight values ​​of anhydrous trehalose and trehalose dihydrate.

[0209] The molar ratios of lipid components in the two optimized formulations, lipid A and lipid B LNP formulations, are shown in Table 2 (CL:cationic lipid).

[0210] [Table 2]

[0211] As shown in Table 3 and Figure 1A, the fold increase in hEPO expression with lipid A and lipid B compared to MC3 indicates that these LNPs are superior to MC3 in delivering mRNA. In the table below, "P2" means PEG2000; "Times MC3" means fold increase relative to MC3; "Std Dev" means standard deviation.

[0212] [Table 3-1] [Table 3-2]

[0213] FIG. 1B shows hEPO expression in mice and non-human primates (NHPs) using LNP lipid A and lipid B. A single dose of hEPO mRNA formulated with lipid A or lipid B (0.1 μg in mice and 10 μg in NHPs) was injected intramuscularly. Serum hEPO levels were quantified at 6, 24, 48, and 72 hours after administration using ELISA. The data show long-term hEPO protein expression in vivo in mice and NHPs for more than 4 days.

[0214] One of the key process parameters identified during optimization is the flow rate during the initial mixing step. Formulations with different final LNP sizes (ranging from 108 to 177 nm) were produced by varying these flow rates during mixing, allowing additional control over process and product attributes. The higher the flow rate, the smaller the particle size. A significant increase in potency occurred when the flow rate reached 375 ml / min, producing an average LNP size of 108 nM. The effect of size on LNP potency was noted as a measure of fold increase in hEPO expression relative to MC3 in Table 4.

[0215] [Table 4]

[0216] The above screening data indicates that the helper lipid DOPE was effective in promoting protein expression. The data also led to the determination of a promising molar composition of four lipids (OF-02 or cKK-E10:DMG-PEG-2K:cholesterol:DOPE=40:1.5:28.5:30). The LNP formulations in 10% trehalose were characterized for all parameters including particle size, PDI, mRNA encapsulation, and mRNA integrity. All tested batches showed the desired characteristics and stability of freeze / thaw cycles. The long-term stability of the formulations at -80°C in 10% (w / v) trehalose was evaluated. The lipid A and lipid B formulations were found to be highly stable. EXAMPLES

[0217] Influenza HIN1 LNP vaccine formulation Influenza pandemics can occur when a novel influenza virus appears in the human population. Such pandemics remain a major threat to public health, requiring vigilance and preparation with protective measures to be used in the event of prolonged human-to-human transmission of the virus. In the experiments described in this example, the hemagglutinin (HA) from the highly pathogenic HIN1 strain A / California / 7 / 2009 (CA09), responsible for the 2009 influenza pandemic, was used as a prototype antigen to evaluate the efficacy of mRNA vaccines produced with lipid A and lipid B LNP formulations.

[0218] HA mRNA was produced as described above. Citrate buffer (1 mM citrate, 150 mM NaCl, pH 4.5) was used in the production of the LNP composition. The citrate buffer containing the mRNA was mixed with lipids in ethanol solution during the formulation process. The pH and concentration of the buffer were selected to achieve a high encapsulation rate of mRNA in the LNP formulation. The two solutions (mRNA in citrate buffer and lipids in ethanol solution) were mixed in a "T" mixer using a pump system to obtain a homogenous pulseless flow, and lipids and mRNA were mixed in a constant ratio throughout the process. This was crucial to achieve a homogenous formulation with the desired size and low PDI, an indicator of a more homogenous size distribution. This method resulted in high mRNA encapsulation, which is crucial to achieve high potency. The solution thus obtained then underwent buffer exchange using TFF / dialysis tubing.

[0219] In mouse studies, the efficacy of lipid A and lipid B CA09 HA formulations was evaluated in a head-to-head comparison with the MC3 LNP formulation as well as recombinant HA (rHA). CA09(H1)HA mRNA (0.4 μg) formulated with different cationic lipids was injected intramuscularly into Balb / C mice (n=8) on day 0 (D0) and day 28 (D28). The immunogenicity of the vaccine, as indicated by the HA inhibition (HAI) titers, is shown in Figure 2A. The data show that dual immunization with lipid A or lipid B on day 0 (D0) and day 28 (D28) induced high HAI titers and allowed complete protection of the animals against a homologous virus challenge (Belgium09 H1N1 virus) (Figure 2B). During the 14-day post-challenge observation period, no overt signs of morbidity (weight loss) were observed in the lipid A and lipid B treatment groups, while a small number of animals in the recombinant protein control group showed morbidity (Figure 2B).

[0220] Similarly, mRNA encoding neuraminidase (NA) from the Mich15 influenza strain (Mich15 N1) was formulated with lipid A and its efficacy was evaluated. Two doses (0.4 or 0.016 μg) of NA mRNA formulated with lipid A were injected intramuscularly into Balb / c mice (n=8). The control group (n=8) was injected with 0.6 μg hEPO mRNA or diluent. Half of the mice received only one injection (1 dose) on study day 0, and the other half received two injections (2 doses) given on study days 0 and 28. The data show that this N1 lipid A formulation elicited a robust immune response as indicated by NA inhibition (NAI) titers (Figure 3A). The data further show that mice treated with one or two doses of the vaccine were protected from a lethal viral challenge with Belgium09 H1N1 (Figure 3B). The level of protection correlated with the NAI titers in vaccine-treated groups versus negative control groups (hEPO and diluent).

[0221] CA09 H1 mRNA formulated with the present LNPs was also tested in the NHP model. mRNA (10 μg) was formulated with lipid A and lipid B and injected intramuscularly into cynomolgus macaques (n=6) on study days 0 and 28. Detectable HAI priming by day 14 and a significant boost in HAI titers by day 28 were observed for all LNPs (Figure 4, right panel). ELISA data also showed significant priming above baseline by day 14 for all doses tested, with a robust boost detected 2 weeks after boost (Figure 4, left panel). The results indicate that the present H1 mRNA formulations generated a robust immune response as shown by HAI and endpoint ELISA titers. EXAMPLES

[0222] Influenza H3N2 LNP vaccine formulation This example describes experiments in which mRNA-LNP vaccine formulations against influenza strain Sing16 (H3N2) are evaluated for efficacy. One of the mRNAs used in these experiments is MRT1400. MRT1400 is a biosynthetic codon-optimized HA-H3 (influenza virus hemagglutinin, H3 subtype) messenger RNA (CO-HA-H3 mRNA) produced by in vitro transcription.

[0223] The protein sequence of influenza virus hemagglutinin, H3 subtype, is shown below. MKTIIALSYI LCLVFAQKIP GNDNSTATLC LGHHAVPNGT IVKTITNDRI EVTNATELVQ NSSIGEICDS PHQILDGENC TLIDALLGDP QCDGFQNKKW DLFVERSKAY SNCYPYDVPD YASLRSLVAS SGTLEFKNES FNWTGVTQNG TSSACIRGSS SSFFSRLNWL THLNYTYPAL NVTMPNKEQF DKLYIWGVHH PGTDKDQIFL YAQSSGRITV STKRSQQAVI PNIGSRPRIR DIPSRISIYW TIVKPGDILL INSTGNLIAP RGYFKIRSGK SSIMRSDAPI GKCKSECITP NGSIPNDKPF QNVNRITYGA CPRYVKHSTL KLATGMRNVP EKQTRGIFGA IAGFIENGWE GMVDGWYGFR HQNSEGRGQA ADLKSTQAAI DQINGKLNRL IGKTNEKFHQ IEKEFSEVEG RVQDLEKYVE DTKIDLWSYN AELLVALENQ HTIDLTDSEM NKLFEKTKKQ LRENAEDMGN GCFKIYHKCD NACIESIRNE TYDHNVYRDE ALNNRFQIKG VELKSGYKDW ILWISFAISC FLLCVALLGF IMWACQKGNI RCNICI* (SEQ ID NO: 1)

[0224] The coding sequence for this protein was codon optimized. The codon-optimized sequence encoding the protein is shown in FIG. 5A (SEQ ID NO:2), and the wild-type sequence is shown as SEQ ID NO:3. The mRNA structure and sequence are shown in FIGS. 5B and 5C, respectively. As shown in the figures, the HA-H3 mRNA coding sequence is flanked by 5' and 3' untranslated regions (UTRs) of 140 and 100 nucleotides, respectively. The biosynthetic HA-H3 mRNA contains a 7-methylguanosine (mGU) residue linked to the first nucleoside of the 5'UTR via an inverted 5'-5' triphosphate bridge. 7It also contains a 5' cap structure consisting of 2'-O-ribose (A) residues, the first nucleoside of which is itself modified by 2'-O-ribose methylation. The 5' cap is essential for the initiation of translation by the ribosome. The entire linear structure is terminated at the 3' end by a tract of approximately 100-500 adenosine nucleosides (polyA). The polyA region is also thought to confer stability to the mRNA and enhance translation. All of these structural elements are naturally occurring components used to promote efficient translation of the HA-H3 mRNA.

[0225] DNA plasmids were constructed to produce codon-optimized mRNA sequences by in vitro transcription. In vitro transcription (IVT) reactions were performed using RNA polymerase. The reaction mixtures were precipitated. The precipitated RNA samples were loaded onto individual depth filtration cassettes, washed with 80% ethanol, and redissolved with recycled H2O. A second aliquot of H2O was pumped out in a manner similar to the first step. This step was repeated once more. The pooled eluates were subjected to ultrafiltration / diafiltration using a 50 kD hollow fiber TFF cassette. Each IVT TFF pool was then diluted in preparation for the cap and tail reaction. The cap-tail reactions were precipitated, and the RNA from the reactions was purified and collected as described above. The filtered mRNA was stored at -20°C until use.

[0226] In these experiments, mRNA encoding Sing16 NA (N2) or Sing16 HA (H3; MRT1400 mRNA) antigens was formulated with lipid A or lipid B and injected intramuscularly into Balb / c mice (n=8) at 0.4 μg mRNA per dose on days 0 and 28. For comparison, 1 μg of recombinant Sing16 H3 or Sing16 N2 protein was injected intramuscularly into Balb / c mice (n=8) with an oil-in-water emulsion adjuvant (AF03). Immune responses were measured by NAI and HAI assays.

[0227] The data show that animals immunized with NA(N2) mRNA demonstrated detectable NAI priming by day 14 and a significant boost in NAI titers by day 28 (Figure 6, right panel). The data also show that HA Sing16 Lipid A and Lipid B formulations elicited robust HAI responses following a boost on day 28 (Figure 6, left panel).

[0228] Similarly, Sing16 HA mRNA lipid A and lipid B vaccines were evaluated in non-human primate (NHP) cynomolgus macaques (n=6). HA Sing16 mRNA (50 μg) formulated with lipid A or lipid B was injected into the monkeys by intramuscular route. The first injection was given on study day 0 and the second injection was given on study day 28. The data show that the vaccine induced a robust immune functional response that was boosted on day 28 (Figure 7A).

[0229] Additionally, four dose levels of HA Sing16 mRNA formulated with lipid A (i.e., MRT5400 vaccine) were evaluated in NHPs: 15, 45, 135, and 250 μg. The first immunization was given on study day 0 and the second immunization on study day 28. All NHPs demonstrated IgG binding and HAI titers for all doses tested, with no differences in immune responses among the various doses tested 2 weeks after the second injection on day 42 (Figures 7B and 7C).

[0230] The Sing16 HA mRNA lipid A vaccine was also evaluated for T cell responses in NHPs after the second vaccination. Peripheral blood mononuclear cells (PBMCs) were collected on day 42 and incubated overnight with Sing16 H3 recombinant protein or a peptide pool representing the entire HA open reading frame. Cytokines induced by restimulation were evaluated by ELISPOT assay. The frequency of PBMCs secreting IFN-γ, a Th1 cytokine (Figure 8A), or IL-13, a Th2 cytokine (Figure 8B), was calculated as spot forming cells (SFC) per million PBMCs. The majority of animals in the three dose level groups tested (250 μg, 135 μg, and 45 μg) showed the presence of a high frequency of IFN-γ-secreting cells, with more than 100 SFC per million PBMCs (Figure 8A). No dose response was observed, as animals in the low and high dose level groups showed comparable frequencies of IFN-γ-secreting cells. In contrast, the presence of IL-13 cytokine-secreting cells was not detected in any of the groups tested at any dose level (Figure 8B). These data provided clear evidence of a Th1-biased cellular response and a lack of Th2 responses to HA antigen following vaccination in NHPs. EXAMPLES

[0231] Influenza LNP vaccine formulations with modified mRNA This example describes an experiment comparing the efficacy of vaccines containing unmodified (unmodified non-replicating or "UNR") and modified (modified non-replicating or "MNR") mRNA. UNR CA09 HA mRNA and MNR CA09 HA mRNA were produced by in vitro transcription. In MNR, all uridines were replaced with pseudouridines.

[0232] Five different doses (0.016, 0.08, 0.4, 2, and 10 μg) of CA09 HA mRNA (modified or unmodified) formulated with lipid A were injected into Balb / c mice (n=15) by intramuscular route. The data show that the LNP formulation increased the stability and delivery efficiency of naked mRNA (UNR), as the potency between UNR and MNR mRNA was comparable as shown by HAI titers (Figure 9A). ELISA data for Balb / c mice also showed significant priming above baseline by day 14 for all doses tested (both UNR and MNR mRNA), with a robust boost detected 2 weeks after the boost. The data also show that UNR and MNR mRNA were comparable in eliciting ELISA titers (Figure 9B).

[0233] In conclusion, this dose titration study demonstrated that unmodified and modified CA09 HA mRNA formulated with lipid A elicited statistically indistinguishable immune responses in Balb / c mice as shown by HAI or by end-point ELISA assay. Balb / c mice immunized with the four higher doses of UNR and MNR mRNA show detectable HAI priming by day 14 and a significant boost in HAI titers by day 42 for all doses. These day 14 priming titers represent both a dose effect and potential dose sparing, producing detectable titers over a 125-fold range. Second injection titers over the same dose range confirm the robustness of the immune response to this mRNA-LNP formulation. Similar results were observed in non-human primates. EXAMPLES

[0234] Multivalent influenza vaccine LNP formulation This example describes a study using a lipid A-based LNP vaccine containing mRNA encoding CA09 HA (described in Example 2) and mRNA encoding Sing16 HA (described in Example 3).

[0235] More specifically, CA09 HA mRNA and Sing16 HA mRNA co-encapsulated in lipid A were evaluated in Balb / c mice (n=8). The mRNA-LNPs were administered as two co-encapsulated mRNAs or administered separately as a single encapsulated mRNA. In both approaches, a total of 0.4 μg of LNP formulation was injected into the mice by intramuscular injection. The first injection was given on study day 0 and the second injection was given on study day 28. The data show that the vaccine elicited a robust immune functional response. There did not appear to be any difference between the two administration approaches. These data show that the co-encapsulation did not cause any disturbance or interference between the two mRNAs. EXAMPLES

[0236] Further studies on multivalent influenza vaccine LNP formulations A panel of unmodified mRNAs encoding CA09 HA, Sing16 HA, Sing16 NA, Mich15 NA, A / Perth / 16 / 2009 influenza virus (Perth09 NA) and reporter antigens of firefly luciferase (FF) and hEPO was prepared.The LNP formulations for HA and NA mRNA-LNP preparations were then tested for in vitro expression, immune response in animals, and efficacy in preclinical models.In this study, all LNP formulations were lipid A formulations.

[0237] material and method mRNA-LNP formulations The mRNA transcripts encoding hEPO, FF, CA09 HA, Sing16 HA, Mich15 NA, and Sing16 NA were synthesized by in vitro transcription with RNA polymerase together with plasmid DNA templates encoding the desired genes using unmodified nucleotides. The purified mRNA precursors thus obtained were further reacted by enzymatic addition of a 5' cap structure (Cap1) and a 3' poly(A) tail of approximately 200 nucleotides in length, determined by gel electrophoresis and purified. All mRNA preparations were analyzed for purity, integrity, and percentage of Cap1 before storage at -20°C. The manufacture of mRNA / lipid nanoparticle (LNP) formulations was described above. Briefly, an ethanolic solution of a mixture of lipids (ionizable lipids, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipids) with a fixed lipid to mRNA ratio was combined with an aqueous buffer solution of target mRNA at acidic pH under controlled conditions to obtain a homogenous suspension of LNPs. The resulting nanoparticle suspension was diluted to final concentration by ultrafiltration and diafiltration into an appropriate diluent system, filtered, and stored frozen at -80°C until use. The mRNA-LNP formulations were characterized for size by dynamic light scattering, percentage encapsulation, and stored at 1 mg / mL at -80°C by dilution with appropriate buffer until further use. hEPO-LNPs and FF-LNPs were utilized to investigate the levels of target protein expression in vivo.

[0238] Visualization of S-protein expressed in HeLa cells Immunocytochemistry-immunofluorescence analysis of influenza NA and HA proteins was performed in HeLa cells transfected with bivalent H3N2 (Sing16 HA and Perth09 NA) mRNA LNPs) using previously described methods (Kalnin et al., npj Vaccines (2021) 6:61). Cells were fixed in 4% paraformaldehyde and targeted antibody staining for HA (GeneTex GTX40258), NA, and the ER marker Calnexin (Abcam ab22595) was performed. Images were captured on a confocal microscope and subsequently imaged for quantification of HA and NA colocalization to the ER, mean signal intensity, and percent cell area.

[0239] Flow cytometry Human skeletal muscle cells (HskMCs, Lonza) were cultured in M199 (Life Technologies) supplemented with GlutaMAX (Life Technologies), streptomycin, penicillin (Gibco), and 20% heat-inactivated FBS (VWR) at 37 °C and 5% CO. Cells were harvested by trypsinization, washed with PBS, and electroporated for 10 min at 4 °C for 1 h according to the manufacturer's electroporation program D-033. 6 12 mg of mRNA per cell was used for electroporation using a human primary muscle cell transfection kit on a Nucleofector 2b (Lonza). After 24 hours, harvested cells were fixed, permeabilized with Cytofix™ / Perm (BD), and stained with CA09 HA (Immune Tech), Sing16 HA (30-2F11-F7-A5, GeneTex), Mich15 NA (6G6, Immune Tech), and Sing16 NA (40017-RP01, Sino Biologicals) specific Abs, followed by PE-conjugated goat anti-mouse IgG secondary Ab (Southern Biotech) or AF647-conjugated goat anti-rabbit IgG (Life Technologies). Antibody-labeled cells were then acquired by Fortessa (BD) and the expression of the respective proteins was analyzed by FlowJo™ (TreeStar).

[0240] Cryo-transmission electron microscope Grids were plasma cleaned prior to LNP sample application using a PELCO easiGlow™ device, and a Vitrobot Mark IV system (ThermoFisher) was used for plunge freezing, with the chamber kept at 100% humidity and 18°C. A 3.0 μl drop of LNP sample was dispensed onto a 300 mesh R2 / 1 QUANTIFOIL® grid with carbon film and gold bars. The grid was blotted for 4 seconds, held in place for 10 seconds, then immediately plunge frozen into liquid ethane for storage and transferred to a Krios microscope. Exposures were collected using a Titan Krios transmission electron microscope (ThermoFisher) equipped with a BioQuantum energy filter and a K3 direct electron detector (Gatan) operated in counting mode. The calibrated physical pixel size on the detector was 1.38 Å, corresponding to a magnification of 64,000. A total of 3,141 69-frame movie exposures were collected with a defocus of -0.5 to -1.7 μm and a dose per frame of 1.045 e / Å2. For each movie exposure, patch-based motion correction, super-resolution pixel binning, and frame weighting were performed using RELION-3.1.34. More than 700 candidate particle coordinates were extracted from the corrected images. Subsequent data analysis was performed using MATLAB R2019a with the Image Processing Toolbox.

[0241] Immunization of mice and NHPs for expression studies Groups of four cynomolgus macaques (NHP) (male and female) and four to eight male BALB / c mice were administered intramuscularly with hEPO-LNPs manufactured at the same ratios intended to be used for the HA / NA mRNA-LNP formulations at a dose of either 10 μg (NHP) or 1, 0.5, 0.1, and 0.05 μg (mouse). Blood samples were taken pre-dose and at 6, 24, 48, 72, and 96 hours post-dose to monitor serum hEPO expression by ELISA using R and D systems, Quantikine® IVD® ELISA, and human erythropoietin immunoassay kits as per manufacturer's protocol and reported as final values ​​in mIU / ml and ng / ml. Briefly, microplate wells pre-coated with a mouse monoclonal antibody specific for EPO were incubated with specimens or standards. After removing excess specimens or standards, the wells were incubated with rabbit anti-EPO polyclonal antibody conjugated to horseradish peroxidase. During the second incubation, the antibody-enzyme conjugate bound to the immobilized EPO. Excess conjugate was removed by washing. A chromogen was added to the wells, which was oxidized by the enzymatic reaction to form a blue complex. The reaction was stopped by adding acid, which changed the blue color to yellow. The amount of color produced was directly proportional to the amount of conjugate bound to the EPO-antibody complex, which in turn was directly proportional to the amount of EPO in the specimen or standard. The absorbance of the complex was measured, and a standard curve was constructed by plotting absorbance versus the concentration of the EPO standard. The EPO concentration of unknown specimens was determined by comparing the optical density of the specimens to the standard curve. The standard used in this assay was recombinant hEPO, a urinary-derived form of human erythropoietin, calibrated against a second international reference (67 / 343).

[0242] Immunization of mice and NHPs for immunogenicity studies Groups of Balb / c mice (Mus musculus) according to treatment group were immunized with a dose of 0.05 mL of the designated vaccine formulation or diluent under isoflurane anesthesia by the IM route in the quadriceps muscle, in one hind limb on day 0 and in the contralateral limb on day 28. Mice that lost more than 20% of their initial body weight and showed significant clinical signs were euthanized prior to study termination after veterinary evaluation of the animal's health.

[0243] Naive male and female crab-eater macaques (Macaca fascicularis) of Mauritius origin were selected for the study. At the start of the study, the animals weighed >2 kg and were >2 years old. Animals selected for the study underwent an extensive physical examination prior to study allocation. Pre-allocation assessment of health status included an on-site veterinary examination and blood sample collection for CBC analysis as applicable per NIRC SOPs. Animals were generally housed in pairs and allowed to acclimate for at least 3 days prior to study initiation. Groups consisted of a maximum of 6 animals per treatment group. All animals were immunized under Ketamine HCl (10 mg / kg, IM) or Telazol (4–8 mg / kg, IM) sedation on study day 0 with a dose of 0.5 ml of their respective vaccine formulation or diluent by the IM route, targeted to the deltoid muscle, in one forelimb of each animal. 28 days after the first immunization was administered, a second immunization was given to the animal's contralateral limb.

[0244] Immunization of mice and NHPs for challenge studies Mice were inoculated with the challenge strain approximately 9–12 weeks after the last immunization. A vial of stock virus was thawed and diluted to the appropriate concentration in ice-cold sterile PBS. All mice were inoculated with 4LD 50 Equivalent to 105.54 TCID in PBS 50Mice were challenged with a total volume of 50 μl containing 1000 mg / kg Belgium09 virus. Virus challenge was performed inside a safety cabinet in an expanded ABSL2 laboratory. Mice were first anesthetized with an IP injection of ketamine / xylazine solution (ketamine 50 mg / kg and xylazine 5 mg / kg) and then challenged IN (droplets in both nostrils; 25 μl per nostril) with influenza virus in a total volume of 50 μl using a micropipette. Following the challenge procedure, mice were placed in supine position and observed until they recovered from anesthesia. Daily body weights were taken following H1N1 challenge. Any individual animal that showed a single observed weight loss of >20% was euthanized. Body weight measurements were recorded in an online database, Pristima® (Version 7.5.0 Build 8), or booked on a study-specific worksheet every day after challenge until euthanasia.

[0245] Blood collection For mice, blood was collected by submandibular or retro-orbital venous plexus bleeding (antemortem bleed, approximately 200 μl before study and on study days 14, 28, and 42) and cardiac puncture (terminal bleed, day 56) from all animals under sedation. Mice were bled before the study to obtain baseline pre-immune serum samples for prescreening purposes. For serum processing, blood samples were collected in SST tubes and allowed to clot for 30 min to 1 h at room temperature. Samples were then centrifuged at 1000-1300 g for 5-10 min with the brake off. Serum was collected using a P200 pipettor, divided into two 0.5 ml cryovials, and stored at -20°C. All bleeds were documented in a specimen collection and processing log, indicating the time of sample collection and the technician responsible for performing the procedure. Portions of serum samples were evaluated in HAI or ELLA and ELISA assays for antibody titers.

[0246] NHPs were bled for serum isolation (days -4, 2, 7, 14, 28, 30, 35, 42, 56, 90, and 180) under anesthesia using ketamine 10 mg / kg / acepromazine 1 mg / kg administered intramuscularly. Blood collection did not exceed established guidelines for percentage of body weight and animal health. Blood was collected from anesthetized NHPs using femoral vein puncture using Vacutainer 21 ga x 1" blood collection needles or Abbott Butterfly 23 ga x 3 / 4" tubing attached to BD Vacutainer® SST™ gel tubes. Serum was isolated by spinning the tubes at a speed of 1200 x g for 10 minutes at room temperature. Serum was then aliquoted into labeled cryovials (1 ml / vial) and stored at ≦-20°C. Portions of serum samples were evaluated in HAI or ELLA and ELISA assays for antibody titers. For PBMC, NHPs were prebled before vaccination and again approximately 42-63 days after the first injection. For this purpose, blood was collected in BD Vacutainer® tubes containing heparin anticoagulant. Briefly, anticoagulated blood samples were diluted in PBS and subjected to gradient density centrifugation at 400×g for 30 min using Histopaque® separation fluid (Sigma). The opaque interface containing mononuclear cells was then collected and washed three times with PBS using low speed (250×g) centrifugation, with a final centrifugation to decount platelets. Live vs. dead PBMC were enumerated using a Nexcelom Cellometer K2. PBMC were cryopreserved in FBS with 10% DMSO using Mr.Frosty® freezing boxes. The boxes were immediately placed in a -80°C freezer for 24 h and then transferred to a liquid nitrogen tank for storage.

[0247] ELISA Antibody ELISA was performed using recombinantly produced Sing16 NA protein, Sing16 HA protein, or CA09 HA protein. Proteins were captured on 96-well high-binding polystyrene plates at a concentration of 2 μg / ml in carbonate-bicarbonate buffer. Plates were harvested and incubated overnight (16 ± 4 h) at 2–8 °C. After overnight incubation, antigen-coated plates were washed five times with wash buffer (PBS, 0.5% Tween 20) and blocked with blocking solution (10% BSA in PBS) for 60 ± 30 min at room temperature. Test samples, naive controls, and reference samples were diluted in sample diluent (PBS 10% BSA 0.5% Tween 20) and added in duplicate to wells, followed by incubation for 90 min at room temperature. Plates were washed five times with wash buffer and goat anti-mouse HRP was added for mouse sera and goat anti-monkey HRP for NHP sera at a dilution of 1:10,000. Plates were then incubated at room temperature for 30 minutes and excess HRP-IgG was washed off with wash buffer. Sure-Blue TMB substrate was added to each plate and the reaction was stopped with TMB stop solution after approximately 10 minutes. Plates were then read at 450 nm using a Thermo Labsystems Multiskan™ spectrophotometer. Anti-antigen (HA or NA) specific antibody titers were expressed as the reciprocal of the highest serum dilution with an absorbance value of >0.3.

[0248] HAI assay HAI assays were performed using Sing16 H3N2 and CA09 H1N1 virus stocks (BIOQUAL, Inc.). Serum was treated with receptor-destroying enzyme (RDE) by diluting 1 part serum with 3 parts enzyme and incubated overnight in a 37°C water bath. The enzyme was inactivated by a 30 min incubation period at 56°C followed by the addition of 6 parts PBS for a final dilution of 1 / 10. HAI assays were performed in V-bottom 96-well plates using 4 hemagglutination units (HAU) of virus and 0.5% turkey RBCs. Reference serum for each strain was included as a positive control on all assay plates. Each plate also included a back titration to verify the antigen dose (4HAU / 25μl) as well as negative control samples (PBS or naive control serum). HAI titers were determined as the highest dilution of serum resulting in complete inhibition of hemagglutination. Results were valid only for plates with appropriate back titration results (demonstrating 4 HAU / 25 μl addition) and reference serum titers within 2-fold of the expected titer.

[0249] NAI assay Neuraminidase inhibition (NAI) antibody titers were determined using a method for enzyme-linked lectin assay (ELLA). The source of antigen (viral NA) was titrated and a standard amount was selected for incubation with serial dilutions of serum. The serum titration was performed using serial dilutions of serum (heat inactivated at 56°C for 1 h) and a standard amount of virus was added in duplicate to wells of fetuin-coated plates. The mixture was then incubated overnight (16-18 h); the following day, HRP-conjugated peanut agglutinin PNA (diluted to 2.5 μg / ml) was added to the washed plates and incubated for 2 h at room temperature. Substrate (ODP in sodium citrate) was added and incubated for 10 min to allow color development. The reaction was then stopped by adding stop buffer (sulfuric acid 1N). Plates were scanned for absorbance at OD490 nm. A reduction or absence of color compared to the virus control indicated that NA activity was inhibited due to the presence of NA-specific antibodies. NAI titer (IC50 IC50 values) were calculated from the OD readings and results were graphed in GraphPad Prism. If the ELLA titration curve did not provide a good enough fit to determine a reliable IC50 value, samples were retested using a different dilution scheme to reach the 50% endpoint.

[0250] T cell ELISPOT assay Complete medium (DMEM1640 + 10% heat inactivated FCS) was pre-warmed in a 37°C water bath. PBMCs were quickly thawed in a 37°C water bath and transferred dropwise into a conical tube with pre-warmed medium. The tubes were centrifuged at 1,500 rpm for 5 min, and the cells were resuspended and counted using a Guava cell counter. Monkey IFN-γ ELISPOT kit (Mabtech 3421M-4APW) and IL-13 ELISPOT kit (Mabtech 3470M-4APW) were used. Pre-coated plates provided by the kit were washed 4 times with sterile PBS and blocked with 200 μl complete medium for at least 30 min in a 37°C incubator. Sing16 H3 peptide pool (Genscript Custom Order) (each peptide at 1 μg / ml) was used as the recall antigen in the assay. ConA (Sigma CAT#C5275) 2 μg / ml was used as a positive control. 50 μl of recall antigen and 300,000 PBMCs in 50 μl were added to each well for stimulation. Plates were placed in a 37° C., 5% CO2 humidified incubator for 48 hours.

[0251] After incubation, cells were removed, plates were washed 5 times with PBS, and 100 μl of 1 μg / ml biotinylated anti-IFN-γ or anti-IL-13 detection antibody was added to each well of the plate. After 2 hours of incubation, plates were washed 5 times with PBS and incubated with 100 μl of 1:1000 dilution of streptavidin in each well for 1 hour at room temperature. Plates were developed with 100 μl of BCIP / NBT substrate solution until spots appeared. Plates were rinsed with tap water, air-dried, scanned, and enumerated using a CTL ImmunoSpot® reader (Cellular Technology Ltd.). Data were reported as spot-forming cells (SFC) per million PBMC.

[0252] Memory B cell (MBC) ELISPOT assay Sing16 H3-specific and total IgG were assayed using a human IgG Single-Color Memory B Cell ELISPOT Kit (CAT#NC1911372, CTL) according to the manufacturer's instructions. + Antibody secreting cells (ASCs) were measured. Differentiation of MBCs to ASCs was performed in PBMCs using the stimulation cocktail provided by the kit. Briefly, frozen PBMCs were quickly thawed in a 37°C water bath, mixed with DNase I (CAT#90083, Fisher Scientific), transferred to a tube containing pre-warmed complete medium (CM) (RPMI 1640, (CAT#22400-089, Gibco) containing 10% FCS (CAT#SH30073.03, HyClone™), and 1% penicillin / streptomycin (CAT#P4333, Sigma) and centrifuged at 1,500 rpm for 5 min. The cell pellet was diluted with 5 ml of complete medium at 2 × 10 per ml. 6The cells were resuspended in PBS and transferred to a T25 flask for 1 h in a 37°C, 5% CO2 incubator. The volume of the cell suspension was then adjusted to 6 ml and B-Poly-S was added at a 1:1000 dilution. The cells were left in the CO2 incubator for stimulation for 4 days. Kit-supplied PVDF microplates were pre-wetted with 70% ethanol, rinsed, and coated overnight with 80 μl / well of kit-supplied anti-human IgG capture Ab or 4 μg / ml of Sing16 / H3 recombinant protein.

[0253] Cells were harvested 4 days after stimulation, washed, counted, and adjusted to the indicated concentrations in CM. Coated microplates were washed with PBS, blocked with CM for 1 hour, and decanted. 100 μl / well of cell suspension was added to the plate and incubated at 37° C. for 18 hours in a CO2 incubator. After washing, 80 μl / well of 1:400 diluted anti-human IgG biotin detection antibody was added to the plate and incubated at room temperature for 2 hours. Following washing, 80 μl / well of 1:1000 diluted streptavidin-AP was added to the plate for 1 hour. Freshly prepared substrate solution was added and incubated at room temperature for 18 minutes. Plates were rinsed with tap water, air-dried, scanned, and enumerated using a CTL ImmunoSpot® reader (Cellular Technology Ltd.). For each individual animal, IgG + The number of antigen-specific ASCs and Sing16 / H3-specific ASCs were calculated per million PBMCs. The frequency of antigen-specific ASCs was calculated based on the total IgG + Calculated as % of antigen-specific ASC to ASC. To assess assay background, negative control wells on all plates were covered with PBS (no background detected).

[0254] statistical analysis Shining T max To estimate T for individual subjects based on observed data, nonparametric methods are used. maxwas estimated. To estimate the half-life of luminosity, a linear model was fitted to the log-transformed data for each subject during the time course of decay from maximum luminosity to baseline, assuming an exponential decay model for luminosity after reaching maximum (we estimate baseline using the mean luminosity in the saline group). The half-life was estimated as the time point at which log luminosity reached the midpoint between maximum and baseline values. For analyses of different readouts with outcomes summarized as geometric means, SE model-based geometric means and SEs were estimated from a mixed-effects model for repeated measures where response was the log-transformed readout, vaccination was a fixed effect, and time was the repeated measure; log-based mean and SE estimates from the model were then transformed back to obtain geometric means and SEs. For weight change, over-descriptive statistical analysis was used. The median and range for each group of maximum % weight loss over time from baseline (day 0) was reported to assess worse-case scenarios; the median and range for each group of % weight change from baseline at last observation was reported to assess weight regain.

[0255] Antigen sequence The sequence of the Perth09 N2 antigen used here is: MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKLAEYRNWSKPQCDITGFAPFSKDNSIRLSAGGDIWVTRE PYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSKEILRTQESECVCINGTCT VVMTDGSASGKADTKILFIEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAFDDGNDVWMGRTISEKSRLGYETFKVIEGWSNPKSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI* (SEQ ID NO: 4) It is.

[0256] The sequence of the Mich15 N1 antigen used here is: MNPNQKIITIGSICMTIGMANLQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIRE PFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSC FTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK* (SEQ ID NO: 5) It is.

[0257] The sequence of the Sing16 H3 antigen used here is: *(SEQ ID NO:6) It is.

[0258] The sequence of the Sing16 N2 antigen used here is: MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVTRE PYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLIDSVVSWSKDILRTQESECVCINGTCT VVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAFDDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADLNLMHI* (SEQ ID NO: 7) It is.

[0259] The sequence of the CA09 H1 antigen used here is: *(SEQ ID NO:24) It is.

[0260] The sequence of the HA strain A / California / 7 / 2009(H1N1)(CA09) antigen mRNA open reading frame (ORF) used here is: It is.

[0261] The sequence of the A / Michigan / 45 / 2015 (Mich15) neuraminidase (NA) antigen mRNA open reading frame (ORF) used here is: It is.

[0262] The sequence of the A / Singapore.INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin antigen mRNA open reading frame (ORF) used here is: It is.

[0263] The sequence of the Perth / 16 / 2009 (H3N2) NA antigen mRNA open reading frame (ORF) used here is: It is.

[0264] The sequence of the A / Wisconsin / 588 / 2019 antigen mRNA open reading frame (ORF) used here is: It is.

[0265] The sequence of the A / Tasmania / 503 / 2020 antigen mRNA open reading frame (ORF) used here is: It is.

[0266] The sequence of the B / Washington / 02 / 2019 antigen mRNA open reading frame (ORF) used here is: It is.

[0267] The sequence of the B / Phuket / 3073 / 2013 antigen mRNA open reading frame (ORF) used here is: It is.

[0268] result mRNA antigen production, characterization, and expression mRNAs encoding full-length codon-optimized HA and NA for various influenza strains were enzymatically synthesized using unmodified ribonucleotides. All mRNA formulations had >95% 5'Cap1 and showed a single homogenous peak on capillary electrophoresis. The mRNA-LNP formulations were produced by mixing various lipid components with mRNA in fixed ratios under controlled conditions. As shown in Table 5, all mRNA-LNPs showed >95% encapsulation, uniform hydrodynamic radii ranging from 95 to 105 nm, and polydispersity index (PDI) ranging from 0.060 to 0.136 (Table 5).

[0269] [Table 5]

[0270] Cryo-electron microscopy (cryo-TEM) images of CA09 HA mRNA-LNPs showed uniform spherical particles with a multi-layered inner core structure. The layered nature of the solid core structure, further analyzed by Fourier transform, showed a periodicity of 3.7 nm between layers. The uniform morphology of the particles seen in the micrographs indicates that the LNPs are homogenous LNP formulations with proper assembly.

[0271] Antigen expression was confirmed by flow cytometry by transiently transfecting human skeletal muscle cells (HskMCs) with non-encapsulated mRNA constructs of CA09 HA, Sing16 HA, Sing16 NA, or Mich15 NA and staining with protein-specific antibodies for analysis. High levels of HA and NA expression from HskMCs were observed, confirming proper assembly and trafficking of native HA trimers and NA tetramers upon expression in muscle cells. To study the subcellular localization of expressed HA and NA proteins, HeLa cells were transfected with bivalent H3N2 LNPs and proteins were visualized by immunostaining and confocal microscopy. Staining of permeabilized cells with antibodies against the corresponding proteins and the endoplasmic reticulum (ER) marker calnexin revealed that NA signals showed strong colocalization in the ER (approximately 90%), whereas HA was moderately colocalized with the ER (25%). This is consistent with the understanding that early NA and HA proteins are trafficked to the ER for assembly (Dou et al., Front Immunol. (2018) 9:1581).

[0272] The efficiency of mRNA delivery by LNPs and the selection of optimal formulation parameters were evaluated using reporter mRNA expression (Thess et al., Molecular Therapy (2015) 23(1):S55). Single doses of either 0.05, 0.1, 1, or 5 μg of unmodified FF-LNP formulations were administered intramuscularly (IM) to mice. Luciferase activity, as measured by mean bioluminescence, peaked at 6 hours post-injection at all doses and was detectable beyond 72 hours, indicating sustained expression from the mRNA construct (Figure 11, panel (a)). High levels of mRNA-mediated protein expression were further validated with hEPO at single doses of 0.1 μg in mice and 10 μg in non-human primates (NHPs). The study was intended to compare LNPs using the standard LNP Dlin-MC3-DMA25 formulation as a control. Serum hEPO, as quantified by ELISA, showed maximal expression at 6 hours, with approximately 12-fold higher erythropoietin expressed using hEPO-LNPs compared to hEPO-MC3 (Figure 11, panel (c)). Both hEPO-LNPs and hEPO-MC3 showed similar expression kinetics in NHPs and were detectable from 6 to 72 hours (Figure 11, panel (d)). These results confirmed the usefulness of this LNP formulation for efficient delivery of mRNA for expression both in vitro and in vivo.

[0273] Immunogenicity of HA (H1, H3) and NA (N1, N2) mRNA-LNPs in mice Natural history and vaccine studies have shown that antibodies against influenza HA and NA have antiviral functions and both antigens are considered important for an effective influenza vaccine (Krammer et al., Nat Rev Immunol. (2019) 19(6):383-97). Unmodified CA09 HA-LNP and Sing16 HA-LNP mRNA vaccines were evaluated in BALB / c mice (n=8) in a two-dose regimen where 2, 0.4, 0.08, or 0.016 μg of mRNA-LNP were administered on a 4-week interval schedule. Total IgG responses were evaluated in ELISA using recombinant HA (rHA) antigens of the same strains. HA-specific antibodies were detected in all groups after one dose, but titers peaked at day 42 after the second dose (Figure 12). To measure functional antibodies, hemagglutination inhibition (HAI) responses were evaluated against the homologous strains CA09 and Sing16. Although HAI titers after the first dose could be observed for the 2 μg dose CA09-LNP and Sing16-LNP treated groups with GMTs of 160 and 70, respectively, on day 28, a more significant increase in HAI titers was observed after the second dose. On day 42, GMT titers were 80 and 2200 for the 0.016 μg and 0.4 μg groups, respectively, for CA09-HA-LNP, and 14 and 100 for the 0.016 μg and 0.4 μg groups, respectively, for the Sing 16 HA-LNP groups (FIG. 13).

[0274] Similarly, to test for anti-NA responses, mice were immunized with 2, 0.4, 0.08, or 0.016 μg of Sing16 NA-LNP or Mich15 NA-LNP. ELISA with recombinant NA antigen was performed to evaluate the total IgG response induced by Mich15 NA-LNP or Sing16 NA-LNP formulations. Animals developed high antibody binding responses after one dose, and there was a significant increase in NA-binding antibodies 42 days after the second dose (Figure 14). Enzyme-linked lectin assay (ELLA) was used as a surrogate of functional antibody titer for neuraminidase inhibitor (NAI) activity against H6N1 or H6N2 chimeric viruses. Although two doses of vaccine substantially increased functional antibody responses when compared to a single dose, robust NAI titers with GMT800 and GMT60 were recorded 28 days after a single dose even at doses as low as 0.016 μg Mich15 NA-LNP and Sing16 NA-LNP, respectively. On day 42, in the Sing16 NA-LNP group, GMT titers between 0.4 μg and 0.016 μg were 900 and 10200, respectively, indicating a dose-dependent response, with titers exceeding the ULOQ for Mich15 NA-LNP (FIG. 15).

[0275] Protection from viral challenge in mice To test the efficacy of mRNA vaccine in a mouse influenza virus challenge model, we IM-vaccinated BALB / c mice with 0.4 μg CA09 HA-LNP along with two doses of LNP dilution buffer in a negative control group on weeks 0 and 4. HAI titers of vaccine group serum samples on study days 0, 14, 28, 42, 56, 92, and 107 indicated a robust immune response with GMTs of 1660 and 1:830 on days 56 and 92, respectively (Figure 16A). On day 93, all mice were inoculated with Belgium09 virus homologous to CA09 at a 4-fold dose (4×LD ) capable of inducing a 50% lethal outcome. 50) intranasally. All vaccine group mice survived the challenge with no mortality and some mild morbidity characterized by a transient weight loss of less than 5% (Figure 16B). However, mice in the diluent control group suffered significant rapid weight loss, which led to a high mortality rate (90%) by day 9. These results demonstrated the high efficacy of the HA-based MRT formulation in a lethal mouse influenza challenge model.

[0276] To evaluate the protective efficacy of NA-based MRT vaccines, we performed a similar challenge experiment in BALB / c mice. Since the Mich15 NA-LNP vaccine induced robust NAI titers after one immunization in naive mice (Figure 16A), we evaluated a one- or two-dose regimen of 0.4 or 0.016 μg Mich15 NA-LNP administered over a 4-week interval. Control groups were vaccinated with the same regimen and received 0.6 μg hEPO-LNP or diluent buffer. Robust NAI titers were observed after one dose, with GMTs of 14,000 NAI at 0.4 μg and 1,800 NAI at 0.016 μg Mich15 NA-LNP recorded on day 28 (Figure 17A). After the second immunization on day 42, NAI titers increased to 108,000 NAI in the 4 μg group and 37,000 NAI in the 0.016 μg group. More than 12 weeks after the vaccination regimen, all groups had 4 × LD 50The mice were challenged with 100 mg of the Belgium09 H1N1 virus. Individual body weight changes over time from baseline by treatment group are shown graphically in FIG. 17B. All mice in the two control groups suffered significant morbidity and all animals had to be euthanized due to >20% body weight loss by day 8 post-infection. Notably, all but one animal in the vaccine group survived the challenge in the single 0.016 μg dose group, indicating high protective efficacy against death even after a single dose of only 0.016 μg Mich15 NA-LNP. A higher dose (0.4 μg) showed high overall protection, but in contrast to HA immunization, NA vaccination was not sufficient to protect against weight loss, as vaccinated animals showed a moderate weight loss of 10% of their initial body weight, consistent with findings reported for other NA vaccines. Weight regain was observed for the vaccinated groups, resulting in a mean final weight change of 2.7% at the low dose and 4.8% at the higher dose compared to baseline. Overall, the results demonstrated that a single low-dose MRT NA-LNP vaccination can induce measurable functional antibodies to block influenza NA activity and sufficient to protect against lethal challenge in mice.

[0277] Immunogenicity of HA(H3)mRNA-LNPs in NHPs To evaluate the immunogenicity of mRNA-LNPs in NHPs, a dose-ranging study was performed in NHPs dealing with 15, 45, 135, and 250 μg of Sing16 HA-LNPs. After the first immunization, all vaccinated NHPs developed antibodies reactive to the recombinant HA protein as noted by ELISA (FIG. 18). A further boost in titers was observed after the second dose. Surprisingly, the 15 μg dose induced ELISA titers that were only 1.8-fold lower than the 135 μg dose level (95% CI 1.0, 3.6), suggesting dose saturation was close to the 15 μg level. Robust HAI antibodies were induced in all dose groups at day 42, with recorded GMTs of 400 for 15 μg, 700 for 45 μg, 900 for 135 μg, and 570 for 250 μg. On day 42, the fold increase in GMT titers with 95% CI was 2.2-fold (1.0; 5.0) for the 135μg to 15μg and 1.3-fold (0.6; 2.8) for the 135μg to 45μg treatment groups, indicating minimal differences between groups despite an observed trend towards higher titers with increasing dose (Figure 19A). Neutralization potency as assessed by microneutralization (MN) assay (Figure 19B) showed a trend towards more of a dose effect, with GMTs of 40 for 15μg, 180 for 45μg, and 300 for 135μg on day 28.

[0278] Because T cells have been shown to be effective in reducing viral load and limiting disease severity in animal models (Rimmelzwaan et al., Vaccine (2008) 26(4):D41-D44; Sridhar et al., Nat Med. (2013) 19(10):1305-12; Sridhar et al., Front Immunol. (2016) 7:195), we assessed recall T cells in NHPs vaccinated with 45, 135, or 250 μg of Sing16 HA-LNP or 45 μg of recombinant HA. PBMCs collected on day 42 were evaluated in IFN-γ (Th1 cytokine) and IL-13 (Th2 cytokine) ELISPOT assays using recall stimulation with pooled overlapping peptides spanning the entire sequence of Sing16 HA. All vaccinated animals except one in the 250 μg group expressed IFN-γ secreting cells, ranging from 28 to 1328 spot forming cells (SFC) per million PBMCs (Figure 20A). Notably, no dose response was observed, with lower and higher dose level groups of animals exhibiting comparable frequencies of IFN-γ secreting cells. In contrast, all animals in the control group immunized with recombinant Sing16 HA protein showed an absence of IFN-γ producing cells. The presence of IL-13 cytokine secreting cells was undetectable or very low in all groups tested (Figure 20B). The data suggest that Sing16 HA-LNPs induced a strong Th1-biased cellular response in NHPs, comparable to the cellular response seen with the SARS-CoV-2 vaccine currently in development, MRT5500 (Kalnin et al., supra).

[0279] To investigate the frequency of memory B cells (MBC) in NHPs after immunization with Sing16 HA-LNPs, an ELISPOT assay was developed to quantify antigen-specific MBC as a readout of humoral immune memory. On day 180, PBMCs were collected from NHPs immunized with Sing16 HA mRNA-LNP 45μg or 15μg formulations or with recombinant HA as a comparator at the 45μg dose. A 4-day polyclonal stimulation of PBMCs, optimized to drive memory B cells to antibody-secreting cells (ASCs), was performed and stimulated PBMCs were plated in antigen-specific ELISPOTs, where the frequency of antigen-specific ASCs could be determined. Antigen-specific memory B cells were then quantified as a percentage of total IgG+ memory B cells. Antigen-specific memory B cells were detected in all animals, with frequencies ranging from 1-5% in the 45μg dose group and 0.3-1.5% in the 15μg dose group. In rHA-immunized animals, memory B cell responses appeared to be significantly lower, as antigen-specific memory B cells were undetectable in five of six animals (Figure 21). We conclude that Sing16 HA-LNP, like other mRNA vaccines, induces a population of anti-HA-specific memory B cells that promises to extend population immunity (Lindgren et al., Front Immunol. (2019) 10:614).

[0280] Multivalent influenza virus antigens An advantage of the mRNA-LNP platform is the flexibility of LNP encapsulation for multiple mRNA antigen constructs. However, this potential needs to be tested to address concerns of antigenic interference. To examine influenza antigen combinations, coencapsulated HA and NA mRNA were formulated into LNPs in H3H1, H3N2, or N1N2 combinations as bivalent formulations containing 0.2 μg of each mRNA, or monovalent containing 0.2 μg of each corresponding antigen. These formulations were administered to mice to determine any antigen interference on immunogenicity by comparing the functional titers of the individual antigens in bivalent versus monovalent formulations (Figure 22, panels (a)-(c) and Table 6).

[0281] [Table 6]

[0282] For the H1H3 combo, no statistically significant difference was found between the coencapsulated and separately administered vaccines for HAI titers at any time point (p=0.2584) and no significant difference was found for H3 titers at day 42 (p=0.8389). For the H3N2 combo, the NA component of the vaccine, in combination with the HA component, elicited high neutralizing antibodies, demonstrating the absence of HA dominance. No statistically significant difference was found between the coencapsulated and separately administered vaccines for H3 titers at any time point (p=0.2960) and no significant difference was found for N2 titers at day 42 (p=0.0904). Similarly, for the N1N2 combo, no statistically significant difference was found for N2 (p=0.3899). N1 titers at day 42 for the coencapsulated and separately administered vaccines were above the limit of quantification. Thus, combinations of N2N1, H3H1, or H3N2 produced equivalent antibody titers to the individual LNPs formulated separately.

[0283] We further investigated tetravalent formulations of co-encapsulated H1, N1, H3, and / or N2 mRNA. These formulations were tested in NHPs at 10 μg total consisting of 2.5 μg of each influenza antigen mRNA and a loading amount of non-coding mRNA (nc mRNA) when required in combination, resulting in tetravalent (H1N1H3N2), bivalent (H1N1 or H3N2), or monovalent (H1, H3, N1, or N2) LNPs (Table 7).

[0284] [Table 7]

[0285] HAI titers against H1 or H3, or NAI titers against N1 or N2 were compared between monovalent versus bivalent or tetravalent formulations (Figure 23). On day 42, HAI titers against H1 in the tetravalent group were comparable when analyzed with those in the H1 monovalent group (p=0.9054, t-test, unpaired, two-tailed) or H1N1 bivalent group (p=0.8002). Similarly, H3 HAI titers in the tetravalent group were comparable when analyzed with those in the H3 monovalent group (p=0.2504) or H3N2 bivalent group (p=0.5894). NAI titers against N1 were nearly identical in groups of animals vaccinated with N1 monovalent mRNA or H1N1 bivalent mRNA or tetravalent H1N1H3N2 mRNA formulations. Similarly, there were no differences in N2 NAI titers between N2 monovalent mRNA (p=0.8485) or H3N2 bivalent mRNA (0.4545) and quadrivalent H1N1H3N2 mRNA formulations.

[0286] Overall, these findings indicate that a HA / NA mRNA-LNP coencapsulated or combination multivalent vaccine at this dose level can deliver all four antigens without concerns about antigenic interference, and all antigens were as immunogenic as in formulations when these antigens were delivered alone. EXAMPLES

[0287] Additional LNP formulations Additional LNP formulations for mRNA vaccines were produced and designated lipid C (containing cationic lipid GL-HEPES-E3-E10-DS-3-E18-1), lipid D (containing cationic lipid GL-HEPES-E3-E12-DS-4-E10), and lipid E ​​(containing cationic lipid GL-HEPES-E3-E12-DS-3-E14). Human erythropoietin (hEPO) mRNA was used as the test mRNA. hEPO expression was measured by ELISA from samples taken from mice injected with LNPs. Samples were taken 6, 24, 48, and 72 hours after injection. As shown in FIG. 24, hEPO expression was consistently higher at all time points for LNP formulations lipid A, lipid B, lipid C, lipid D, and lipid E ​​compared to the control LNP formulation containing cationic lipid MC3.

[0288] Table 8 below summarizes the results compared to control LNPs containing MC3 cationic lipid.

[0289] [Table 8]

[0290] The same hEPO mRNA-LNP formulations were then tested in non-human primates (NHPs). Samples were taken 6, 48, and 96 hours after injection. As shown in Figure 25, each LNP formulation produced levels of hEPO comparable to the MC3 control formulation.

[0291] Influenza HA-encoding mRNA-LNP formulations were also tested in NHPs. NHPs were administered the LNP formulations at 10 μg by intramuscular injection, and samples were taken 28 and 42 days after injection. HAI titers were measured as described above. As shown in Figure 26, each LNP formulation produced HAI titers comparable to or higher than the MC3 control formulation.

[0292] The same experiment as shown in Figure 26 was performed and HAI titers were measured using Cal09 H1 influenza antigen. As shown in Figure 27, each of the LNP formulations produced HAI titers comparable to or higher than the MC3 control formulation.

[0293] As shown in FIG. 28, HAI titers using Sing16 H3 antigen were increased with LNP formulations lipid C and lipid D. EXAMPLES

[0294] Further studies on quadrivalent or octavalent influenza vaccine LNP formulations HAI and NAI titers were measured from mice administered the various multivalent LNP-influenza mRNA vaccines. HAI titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / Maryland / 15 / 2016 BX69A, and B / Phuket / 3073 / 2013. NAI titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / Colorado / 06 / 201, and B / Phuket / 3073 / 2013.

[0295] HAI and NAI titers were compared for mice receiving monovalent or quadrivalent HA or NA mRNA vaccines.

[0296] Mice were injected with a prime vaccine on day 0 and the same dose of a booster vaccine on day 21. Blood was collected on days 1, 20, 22, and 35. Monovalent compositions containing mRNA encoding HA or NA antigens used mRNA encoding each of the following individually: H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013). A tetravalent vaccine composition was also prepared containing mRNA encoding N1, N2, NA from the B / Victoria lineage and NA from the B / Yamagata lineage, as well as H1, H3, HA from the B / Victoria lineage and HA from the B / Yamagata lineage (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013). Finally, an octavalent vaccine composition containing mRNAs encoding H1, H3, HA, HA, N1, N2, NA, and NA from the B / Victoria lineage (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013) was prepared and administered as an octavalent vaccine. For all compositions, each mRNA was added in an amount of 0.4 μg / strain. For each group, n=6 mice.

[0297] A summary of each experimental group is listed in Table 9 below.

[0298] [Table 9]

[0299] As shown in Figure 29, the octavalent mRNA-LNP formulation produced HAI titers within 4-fold of the tetravalent for three of the four influenza strains.

[0300] A summary of the NAI titer results for each of the above groups is shown in Figure 31. The octavalent mRNA-LNP formulation produced NAI titers comparable to the tetravalent mRNA-LNP formulation.

[0301] Thus, the data demonstrate that the octavalent vaccine was able to induce robust HA and NA immune responses, and that the presence of immunodominant HAs from four different influenza strains does not appear to suppress or interfere with anti-NA immune responses.

[0302] High content imaging-based neutralization test (HINT) titers for HA and NAI titers were further measured from ferrets administered various multivalent LNP-influenza mRNA vaccines. The HINT assay is described in more detail in Jorquera et al. (Scientific Reports. 9:2676. 2019), which is incorporated herein by reference. HINT titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / IOWA / 06 / 2017, and B / Phuket / 3073 / 2013. NAI titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / Colorado / 06 / 2017, and B / Phuket / 3073 / 2013.

[0303] Ferrets used to evaluate polyvalent vaccine immunogenicity were vaccinated twice, 21 days apart, with (1) a mixture of four mRNAs encoding NA antigens (N1, N2, BvNA, and ByNA), (2) a mixture of four mRNAs encoding HA antigens (H1, H3, BvHA, and ByHA), or (3) a mixture of four mRNAs encoding NA antigens (N1, N2, BvNA, and ByNA) and four mRNAs encoding HA antigens (H1, H3, BvHA, and ByHA), as shown in Table 12 below. Each HA contained HA from one of the following four strains: A / Michigan / 45 / 2015 (H1); A / Singapore / Infimh-16-0019 / 2016 (H3); B / Iowa / 06 / 2017 (B / Victoria lineage); and B / Phuket / 3073 / 2013 (B / Yamagata lineage). All antigens were administered in a 1:1 ratio.

[0304] A summary of each experimental group is listed in Table 10 below.

[0305] All ferrets were bled under sedation (isoflurane) at baseline, 1 day or immediately prior to the booster, at the time of booster vaccination, and 2 weeks post-challenge as required. Serum samples (stored at -20°C until required) were tested by ELLA to assess NAI activity. In addition, a hemagglutination inhibition assay (HAI) was undertaken to assess antibody responses to hemagglutinin antigens following polyvalent vaccination.

[0306] [Table 10]

[0307] A summary of the HINT results for each of the above groups is shown in Figure 30. The octavalent mRNA-LNP formulation produced HINT titers comparable to the tetravalent mRNA-LNP formulation.

[0308] A summary of the NAI titer results for each of the above groups is shown in Figure 32 (day 20) and Figure 33 (day 42). The octavalent mRNA-LNP formulations produced NAI titers comparable to the tetravalent mRNA-LNP formulations. This was true for day 20 through day 42 samples.

Claims

**Claim 1** An influenza vaccine composition comprising eight messenger RNAs (mRNAs), each mRNA comprising an open reading frame (ORF) encoding a different influenza antigen. **Claim 2** The influenza vaccine composition according to claim 1, comprising eight mRNAs encoding (i) one or more hemagglutinin (HA) antigens, (ii) one or more neuraminidase (NA) antigens, or (iii) at least one HA antigen and at least one NA antigen. **Claim 3** The influenza vaccine composition according to claim 1, comprising one or more mRNAs encoding antigens of influenza A, B, and / or C viruses, the antigens being HA and / or NA antigens of influenza A and influenza B viruses. **Claim 4** The antigen is an HA and / or NA antigen of influenza A and influenza B viruses, and optionally, the HA antigen of influenza A virus is selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18; the NA antigen of influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11; the HA and NA antigens of influenza B virus are from the influenza B / Yamagata lineage or the influenza B / Victoria lineage; and / or the HA antigen and the NA antigen are selected from the group consisting of subtypes H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H10N7 and / or lineages B / Yamagata and B / Victoria, the influenza vaccine composition according to claim 1. **Claim 5** The influenza vaccine composition according to claim 1, comprising one mRNA encoding an H3 HA antigen, one mRNA encoding an H1 HA antigen, one mR NA encoding an HA antigen from the influenza B / Yamagata lineage, and one mRNA encoding an HA antigen from influenza B / Victoria. **Claim 6** The influenza vaccine composition according to claim 1, comprising one mRNA encoding the H3 HA antigen, one mRNA encoding the N2 NA antigen, one mRNA encoding the H1 HA antigen, one mRNA encoding the N1 NA antigen, one mRNA encoding the HA antigen derived from the influenza B / Yamagata lineage, one mRNA encoding the NA antigen derived from the influenza B / Yamagata lineage, one mRNA encoding the HA antigen derived from the influenza B / Victoria lineage, and one mRNA encoding the NA antigen derived from the influenza B / Victoria lineage.

7. The ORF is codon-optimized; The mRNA molecule comprises at least one 5'untranslated region (5'UTR), at least one 3'untranslated region (3'UTR), and at least one polyadenylation (poly(A)) sequence; and / or The mRNA comprises at least one chemical modification, the influenza vaccine composition according to claim 1.

8. At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified; and / or At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified, the influenza vaccine composition according to claim 1.

9. The influenza vaccine composition according to claim 7, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

10. The influenza vaccine composition according to claim 1, wherein the mRNA is formulated in lipid nanoparticles (LNP).

11. The LNP contains at least one cationic lipid, optionally the cationic lipid is biodegradable or non-biodegradable; and / or the cationic lipid is cleavable or non-cleavable, the influenza vaccine composition according to claim 10.

12. The influenza vaccine composition according to claim 11, wherein the cationic lipid is selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14.

13. The influenza vaccine composition according to claim 10, wherein the LNP further comprises a polyethylene glycol (PEG) conjugate (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

14. The LNP contains the cationic lipid in a molar ratio of 35% to 55%; the polyethylene glycol (PEG) conjugate (PEGylated) lipid in a molar ratio of 0.25% to 2.75%; the cholesterol-based lipid in a molar ratio of 20% to 45%; and the helper lipid in a molar ratio of 5% to 35% wherein all molar ratios are based on the total lipid content of the LNP, the influenza vaccine composition according to claim 10.

15. The LNP contains the cationic lipid in a molar ratio of 40%; the PEGylated lipid in a molar ratio of 1.5%; cholesterol-based lipid in a molar ratio of 28.5%; and helper lipid in a molar ratio of 30% The influenza vaccine composition according to claim 14, comprising.

16. The influenza vaccine composition according to claim 13, wherein the pegylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

17. The influenza vaccine composition according to claim 13, wherein the cholesterol-based lipid is cholesterol.

18. The influenza vaccine composition according to claim 13, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

19. The LNP is a cationic lipid selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 40%; DMG-PEG2000 in a molar ratio of 1.5%; cholesterol in a molar ratio of 28.5%; and DOPE in a molar ratio of 30% The influenza vaccine composition according to claim 13, comprising.

20. The influenza vaccine composition according to the claim, wherein the LNP has an average diameter of 30 nm to 200 nm or 80 nm to 150 nm.

21. The influenza vaccine composition according to claim 10, comprising 1 mg / mL to 10 mg / mL of LNP.

22. The influenza vaccine composition according to claim 10, wherein the LNP contains 1 to 20 mRNA molecules, and optionally, the LNP contains 5 to 10 or 6 to 8 mRNA molecules.

23. The influenza vaccine composition according to claim 10, wherein the LNP contains two or more mRNAs, and each mRNA encodes a different influenza antigen.

24. The influenza vaccine composition according to claim 10, wherein the composition contains 8 LNPs, and each LNP contains an mRNA encoding a different influenza antigen.

25. The influenza vaccine composition according to claim 1, formulated for intramuscular injection and optionally containing phosphate-buffered saline.

26. A pharmaceutical composition for use in a method of inducing an immune response in a subject in need thereof, the method comprising administering a prophylactically effective amount of the influenza vaccine composition according to any one of claims 1 to 25 to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

27. A pharmaceutical composition for use in a method of preventing influenza infection or reducing one or more symptoms of influenza infection, the method comprising administering a prophylactically effective amount of the influenza vaccine composition according to any one of claims 1 to 25 to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, and optionally the influenza vaccine composition induces an immune response against one or more seasonal and / or pandemic influenza strains.

28. Administering to a subject one or more doses of an influenza vaccine composition, each dose containing from about 1 μg to about 250 μg of mRNA, optionally each dose containing about 2.5, 5, 15, 45, or 135 μg of mRNA; and / or The pharmaceutical composition according to claim 26, comprising administering two doses of the influenza vaccine composition to the subject at intervals of 2 to 6 weeks, optionally 4 weeks.

29. Use of the influenza vaccine composition according to any one of claims 1 to 25 for the manufacture of a medicament for use in treating a subject in need thereof.

30. The influenza vaccine composition according to any one of claims 1 to 25 for use in treating a subject in need thereof.

31. A kit comprising a container containing a single-use or multiple-use dosage of the composition according to any one of claims 1 to 25, optionally the container is a vial or a prefilled syringe or injector.

32. The influenza vaccine composition according to any one of claims 1 to 25, wherein the influenza antigen comprises an influenza virus HA antigen and / or an influenza virus NA antigen having a molecular sequence identified or designed from a machine learning model.

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